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1
Costanzo, Michele, Armando Cevenini, Emanuela Marchese, Esther Imperlini, Maddalena Raia, Luigi Del Vecchio, Marianna Caterino, and Margherita Ruoppolo. "Label-Free Quantitative Proteomics in a Methylmalonyl-CoA Mutase-Silenced Neuroblastoma Cell Line." International Journal of Molecular Sciences 19, no. 11 (November 13, 2018): 3580. http://dx.doi.org/10.3390/ijms19113580.
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Methylmalonic acidemias (MMAs) are inborn errors of metabolism due to the deficient activity of methylmalonyl-CoA mutase (MUT). MUT catalyzes the formation of succinyl-CoA from methylmalonyl-CoA, produced from propionyl-CoA catabolism and derived from odd chain fatty acids β-oxidation, cholesterol, and branched-chain amino acids degradation. Increased methylmalonyl-CoA levels allow for the presymptomatic diagnosis of the disease, even though no approved therapies exist. MMA patients show hyperammonemia, ketoacidosis, lethargy, respiratory distress, cognitive impairment, and hepatomegaly. The long-term consequences concern neurologic damage and terminal kidney failure, with little chance of survival. The cellular pathways affected by MUT deficiency were investigated using a quantitative proteomics approach on a cellular model of MUT knockdown. Currently, a consistent reduction of the MUT protein expression was obtained in the neuroblastoma cell line (SH-SY5Y) by using small-interfering RNA (siRNA) directed against an MUT transcript (MUT siRNA). The MUT absence did not affect the cell viability and apoptotic process in SH-SY5Y. In the present study, we evaluate and quantify the alterations in the protein expression profile as a consequence of MUT-silencing by a mass spectrometry-based label-free quantitative analysis, using two different quantitative strategies. Both quantitative methods allowed us to observe that the expression of the proteins involved in mitochondrial oxido-reductive homeostasis balance was affected by MUT deficiency. The alterated functional mitochondrial activity was observed in siRNA_MUT cells cultured with a propionate-supplemented medium. Finally, alterations in the levels of proteins involved in the metabolic pathways, like carbohydrate metabolism and lipid metabolism, were found.
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Stewart, Gavin S., Robert A. Fenton, Weidong Wang, Tae-Hwan Kwon, Stanley J. White, Valerie M. Collins, Gordon Cooper, Søren Nielsen, and Craig P. Smith. "The basolateral expression of mUT-A3 in the mouse kidney." American Journal of Physiology-Renal Physiology 286, no. 5 (May 2004): F979—F987. http://dx.doi.org/10.1152/ajprenal.00334.2003.
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Facilitative UT-A urea transporters play a central role in the urinary concentrating mechanism. There are three major UT-A isoforms found in the mouse kidney: mUT-A1, mUT-A2, and mUT-A3. The major aim of this study was to identify the location and function of mUT-A3. UT-A proteins were investigated using three novel mouse UT-A-targeted antibodies: ML446, MQ2, and ML194. ML446 detected mUT-A1 and mUT-A3. ML194 detected mUT-A1 and mUT-A2. Importantly, MQ2 was found to be selective for mUT-A3. MQ2 detected a 45- to 65-kDa signal in the mouse kidney inner medulla, which was deglycosylated to a 40-kDa protein band. Immunolocalization studies showed that mUT-A3 was strongly detected in the papillary tip, mainly in the basolateral regions of inner medullary collecting duct (IMCD) cells. Immunoblotting of subcellular fractions of inner medullary protein suggested that in mouse kidney mUT-A3 was present in plasma membranes. Consistent with this, immunoelectron microscopy demonstrated that mUT-A3 was predominantly localized at the basal plasma membrane domains of the IMCD cells in mouse kidney. Heterologous expression of mUT-A3-enhanced green fluorescent protein in Madin-Darby canine kidney cells showed that the protein localized to the basolateral membrane. In conclusion, our study indicates that mUT-A3 is a basolateral membrane transporter expressed in IMCD cells.
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Lewintre, Eloisa Jantus, Miguel Marin, Cristina Reinoso, David Montaner, Joaquín Dopazo, Juan Jose Calvete, and Javier Garcia-Conde. "Analysis of B-CLL Transcriptomic and Proteomic Profiles: Differences between Molecular Subgroups." Blood 108, no. 11 (November 1, 2006): 2088. http://dx.doi.org/10.1182/blood.v108.11.2088.2088.
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Abstract INTRODUCTION: B cell chronic lymphocytic leukemia (B-CLL) is a lymphoproliferative disorder with a variable clinical course. Patients (pts) with unmutated (unmut) IgVH gene show a shorter progression free survival and overall survival than the patients with IgVH mutated (mut). To understand the differences between molecular subgroups of B-CLL we have studied transcriptomic and proteomic profiles on samples from 40 B-CLL pts (Binet stage A). MATERIAL AND METHODS: 100 μg of total PBMC proteins were used for IEF followed by 2D electrophoresis. Image analysis of scanned gels was used to identify statistically differentially expressed proteins. Image acquisition and analysis were performed using the Ludesi software (http://www.ludesi.com). Selected spots were subjected to automatic digestion and the proteins were identified by MALDI-TOF (Voyager DE-Pro, Applied Biosystems) peptide mass fingerprint using the Protein Prospector software. To confirm the initial results, sequencing of selected peptide ions was carried out by collision-induced dissociation (CID) with a nESI-QTRAP mass spectrometer from Applied Biosystems. Eight proteins were validated by Western Blot. Total RNA from B cells was used to analyze the expression profile by hybridization with Whole Human Genome U133 Plus 2.0 Array from Affymetrix. Normalization, differential gene expression and functional annotations were analyzed using the GEPAS suite (http://www.gepas.org). qPCR using TaqMan primers/probes was used for validation of the microarray data RESULTS: When we compared IgVH mut vs unmut transcriptomic and proteomic profiles, we found more than 600 differentially expressed genes and 12 proteins ( fdr <0.05 adjusted for multiple test contrast and p<0.05, Mann-Whitney’s test, for gene and proteins, respectively). In tables 1 and 2 we show some of the most differentially expressed gene/proteins in each group of pts. Validation of results from microarrays and proteomic data using qPCR and Western blot are in progress. We obtained positive correlation between transcriptomic and proteomic profiles, (corr=0.21, p=0.04, Pearson’s correation test) suggesting that common features are found using both approximations. CONCLUSION: We found a number of interesting gene/proteins that could be able to differentiate molecular subgroups of B-CLL pts. The study of these proteins and genes may lead to better understand the different clinical behaviour of IgVH mut and unmut B-CLL forms, but validation with a larger group of pts is still necessary. Table 1: Genes differentially expressed IgVH mut IgVH unmut Genes annotated using their gene symbol BCL11A MGC9913 DUSP22 RGS4 PDLIM5 CRY1 RDH13 GGT2 PHF15 DMD SVH TUBB6 ADAM29 LPL ITPKB ITGA9 RBKS BCL7A NFATC1 MYEOV RIN3 PPP1R9A Table 2: Proteins differentially expressed IgVH mut IgVH unmut Proteins were annotated according to their gene symbol VIM ERP29 COTL1 CCT2 S100A9 PSMB10 HSPD1
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Pitocchi, Rossana, Paola Cicatiello, Leila Birolo, Alessandra Piscitelli, Elena Bovio, Giovanna Cristina Varese, and Paola Giardina. "Cerato-Platanins from Marine Fungi as Effective Protein Biosurfactants and Bioemulsifiers." International Journal of Molecular Sciences 21, no. 8 (April 21, 2020): 2913. http://dx.doi.org/10.3390/ijms21082913.
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Two fungal strains, Aspergillus terreus MUT 271 and Trichoderma harzianum MUT 290, isolated from a Mediterranean marine site chronically pervaded by oil spills, can use crude oil as sole carbon source. Herein, these strains were investigated as producers of biosurfactants, apt to solubilize organic molecules as a preliminary step to metabolize them. Both fungi secreted low molecular weight proteins identified as cerato-platanins, small, conserved, hydrophobic proteins, included among the fungal surface-active proteins. Both proteins were able to stabilize emulsions, and their capacity was comparable to that of other biosurfactant proteins and to commercially available surfactants. Moreover, the cerato-platanin from T. harzianum was able to lower the surface tension value to a larger extent than the similar protein from A. terreus and other amphiphilic proteins from fungi. Both cerato-platanins were able to make hydrophilic a hydrophobic surface, such as hydrophobins, and to form a stable layer, not removable even after surface washing. To the best of our knowledge, the ability of cerato-platanins to work both as biosurfactant and bioemulsifier is herein demonstrated for the first time.
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Costanzo, Michele, Marianna Caterino, Armando Cevenini, Vincent Jung, Cerina Chhuon, Joanna Lipecka, Roberta Fedele, Ida Chiara Guerrera, and Margherita Ruoppolo. "Proteomics Reveals that Methylmalonyl-CoA Mutase Modulates Cell Architecture and Increases Susceptibility to Stress." International Journal of Molecular Sciences 21, no. 14 (July 15, 2020): 4998. http://dx.doi.org/10.3390/ijms21144998.
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Methylmalonic acidemia (MMA) is a rare inborn error of metabolism caused by deficiency of the methylmalonyl-CoA mutase (MUT) enzyme. Downstream MUT deficiency, methylmalonic acid accumulates together with toxic metabolites from propionyl-CoA and other compounds upstream of the block in the enzyme pathway. The presentation is with life-threatening acidosis, respiratory distress, brain disturbance, hyperammonemia, and ketosis. Survivors develop poorly understood multi-organ damage, notably to the brain and kidneys. The HEK 293 cell line was engineered by CRISPR/Cas9 technology to knock out the MUT gene (MUT-KO). Shotgun label-free quantitative proteomics and bioinformatics analyses revealed potential damaging biological processes in MUT-deficient cells. MUT-KO induced alteration of cellular architecture and morphology, and ROS overproduction. We found the alteration of proteins involved in cytoskeleton and cell adhesion organization, cell trafficking, mitochondrial, and oxidative processes, as validated by the regulation of VIM, EXT2, SDC2, FN1, GLUL, and CHD1. Additionally, a cell model of MUT-rescuing was developed in order to control the specificity of MUT-KO effects. Globally, the proteomic landscape of MUT-KO suggests the cell model to have an increased susceptibility to propionate- and H2O2-induced stress through an impairment of the mitochondrial functionality and unbalances in the oxidation-reduction processes.
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Samodra, Eunike Marganingrum Andriani, Dian Suroto, Tyas Utami, Rachma Wikandari, and Endang Sutriswati Rahayu. "Cold Stress Response Genes of Lantiplantibacillus plantarum subsp. plantarum Mut-3 and Lantiplantibacillus plantarum subsp. plantarum Mut-7 Support the Ability to Survive in Low-Temperature Conditions." HAYATI Journal of Biosciences 30, no. 1 (August 19, 2022): 65–70. http://dx.doi.org/10.4308/hjb.30.1.65-70.
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Probiotics are widely consumed in various food matrices to provide health benefits to the host. The viability of probiotic cells is influenced by several factors, including exposure to high temperatures during the production process and low temperatures during storage. In this study, we report the response to cold stress of Lantiplantibacillus plantarum subsp. plantarum Mut-3 and Mut-7 after 24 h of storage at 4°C and -20℃. The cell number of Lantiplantibacillus plantarum subsp. plantarum Mut-3 and Mut-7 in low-temperature condition had no significant differences than their initial number: 11.88 log CFU/ml and 11.62 log CFU/ml at 4°C; 11.51 log CFU/ml and 11.47 log CFU/ml at -20°C for Mut-3 and Mut-7 respectively. The results indicated the survival capacity of Lantiplantibacillus plantarum subsp. plantarum Mut-3 and Mut-7 at low temperatures. The genes encoding cold shock proteins for the response to cold stress were evaluated by genome sequencing. The CspA/CspC genes of Lantiplantibacillus plantarum subsp. plantarum Mut-3 and Mut-7 possibly play a role in maintaining cell resistance at low temperatures, since the genes products predicted to have conserved motifs in the RNA binding protein (RNP) -1 and RNP-2 responsible for cold response stress which are similar to those in other bacteria.
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Chinsangaram, Jarasvech, Clayton Beard, Peter W. Mason, Marla K. Zellner, Gordon Ward, and Marvin J. Grubman. "Antibody Response in Mice Inoculated with DNA Expressing Foot-and-Mouth Disease Virus Capsid Proteins." Journal of Virology 72, no. 5 (May 1, 1998): 4454–57. http://dx.doi.org/10.1128/jvi.72.5.4454-4457.1998.
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ABSTRACT Candidate foot-and-mouth disease (FMD) DNA vaccines designed to produce viral capsids lacking infectious viral nucleic acid were evaluated. Plasmid DNAs containing a portion of the FMDV genome coding for the capsid precursor protein (P1-2A) and wild-type or mutant viral proteinase 3C (plasmids P12X3C or P12X3C-mut, respectively) were constructed. Cell-free translation reactions programmed with pP12X3C (wild-type 3C) and pP12X3C-mut produced a capsid precursor, but only the reactions programmed with the plasmid encoding the functional proteinase resulted in P1-2A processing and capsid formation. Baby hamster kidney (BHK) cells also produced viral capsid proteins when transfected with these plasmids. Plasmid P12X3C was administered to mice by intramuscular, intradermal, and epithelial (gene gun) inoculations. Anti-FMD virus (FMDV) antibodies were detected by radioimmunoprecipitation (RIP) and plaque reduction neutralization assays only in sera of mice inoculated by using a gene gun. When pP12X3C and pP12X3C-mut were inoculated into mice by using a gene gun, both plasmids elicited an antibody response detectable by RIP but only pP12X3C elicited a neutralizing antibody response. These results suggest that capsid formation in situ is required for effective immunization. Expression and stimulation of an immune response was enhanced by addition of an intron sequence upstream of the coding region, while addition of the FMDV internal ribosome entry site or leader proteinase (L) coding region either had no effect or reduced the immune response.
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Capaci, Valeria, Fiamma Mantovani, and Giannino Del Sal. "A mutant p53/Hif1α/miR-30d axis reprograms the secretory pathway promoting the release of a prometastatic secretome." Cell Stress 4, no. 11 (November 9, 2020): 261–64. http://dx.doi.org/10.15698/cst2020.11.235.
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TP53 missense mutations are frequent driver events during tumorigenesis. The majority of TP53 mutations are missense and occur within the DNA binding domain of p53, leading to expression of mutant p53 (mut-p53) proteins that not only lose the tumor suppressive functions of the wild-type (wt-p53) form, but can also acquire novel oncogenic features fostering tumor growth, metastasis and chemoresistance. Mut-p53 affects fundamental cellular pathways and functions through different mechanisms, a major one being the alteration of gene expression. In our recent work (Capaci et al., 2020, Nat Commun) we found that mut-p53, via miR-30d, modifies structure and function of the Golgi apparatus (GA) and induces increased rate of trafficking. This culminates in the release of a pro-malignant secretome, which is capable of remodeling the tumor microenvironment (TME), to increase stiffness of the extracellular matrix (ECM), favouring metastatic colonization, as shown by cell-based assays and experiments of metastatic niche preconditioning in mouse xenograft models. This study provides new insights into the mechanisms by which mut-p53, through induction of non-coding RNAs, can exert pro-tumorigenic functions in a non-cell-autonomous fashion, and highlights potential non-invasive biomarkers and therapeutic targets to treat tumors harboring mut-p53 (Figure 1).
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Tian, Bing, Yuhong Zhang, Bruce A. Luxon, Roberto P. Garofalo, Antonella Casola, Mala Sinha, and Allan R. Brasier. "Identification of NF-κB-Dependent Gene Networks in Respiratory Syncytial Virus-Infected Cells." Journal of Virology 76, no. 13 (July 1, 2002): 6800–6814. http://dx.doi.org/10.1128/jvi.76.13.6800-6814.2002.
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ABSTRACT Respiratory syncytial virus (RSV) is a mucosa-restricted virus that is a leading cause of epidemic respiratory tract infections in children. In epithelial cells, RSV replication activates nuclear translocation of the inducible transcription factor nuclear factor κB (NF-κB) through proteolysis of its cytoplasmic inhibitor, IκB. In spite of a putative role in mediating virus-inducible gene expression, the spectrum of NF-κB-dependent genes induced by RSV infection has not yet been determined. To address this, we developed a tightly regulated cell system expressing a nondegradable, epitope-tagged IκBα isoform (Flag-IκBα Mut) whose expression could be controlled by exogenous addition of nontoxic concentrations of doxycycline. Flag-IκBα Mut expression potently inhibited IκBα proteolysis, NF-κB binding, and NF-κB-dependent gene transcription in cells stimulated with the prototypical NF-κB-activating cytokine tumor necrosis factor alpha (TNF-α) and in response to RSV infection. High-density oligonucleotide microarrays were then used to profile constitutive and RSV-induced gene expression in the absence or presence of Flag-IκBα Mut. Comparison of these profiles revealed 380 genes whose expression was significantly changed by the dominant-negative NF-κB. Of these, 236 genes were constitutive (not RSV regulated), and surprisingly, only 144 genes were RSV regulated, representing numerically ∼10% of the total population of RSV-inducible genes at this time point. Hierarchical clustering of the 144 RSV- and Flag-IκBα Mut-regulated genes identified two discrete gene clusters. The first group had high constitutive expression, and its expression levels fell in response to RSV infection. In this group, constitutive mRNA expression was increased by Flag-IκBα Mut expression, and the RSV-induced decrease in expression was partly inhibited. In the second group, constitutive expression was very low (or undetectable) and, after RSV infection, expression levels strongly increased. In this group, NF-κB was required for RSV-inducible expression because Flag-IκBα Mut expression blocked their induction by RSV. This latter cluster includes chemokines, transcriptional regulators, intracellular proteins regulating translation and proteolysis, and secreted proteins (complement components and growth factor regulators). These data suggest that NF-κB action induces global cellular responses after viral infection.
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Dal Bo, Michele, Federico Pozzo, Riccardo Bomben, Antonella Zucchetto, Erika Tissino, Dania Benedetti, Massimo Degan, et al. "Nucleophosmin-1 and Ribosome-Associated Components Are Constitutively Overexpressed in NOTCH1 Mutated IGHV Unmutated CLL." Blood 120, no. 21 (November 16, 2012): 3880. http://dx.doi.org/10.1182/blood.v120.21.3880.3880.
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Abstract Abstract 3880 Background: activating mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM) CLL, chemorefractory CLL and CLL in advanced disease phases. In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain and cause an accumulation of an active NOTCH1 isoform. Notably, about 80% of NOTCH1 mutations are represented by a CT frameshift deletion at nucleotides 7544–7545 (c.7544–7545delCT). Clinically, the presence of NOTCH1 mutations is an independent predictor of overall survival in CLL and identifies a subset of patients with particularly unfavourable prognosis (Rossi et al, Blood, 119, 521, 2012). Aim: to identify peculiar molecular and biological features of NOTCH1 mutated CLL in the context of IGHV-UM CLL. Methods: the presence of the c.7544–7545delCT NOTCH1 frameshift deletion was investigated by an ad-hoc amplification refractory mutation system (ARMS) PCR set up to obtain an amplicon specific for the NOTCH1 mutated form and a second amplicon as control. The percentage of NOTCH1 DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR), calculating the ratio between the amount of the specific NOTCH1 mutated amplicon and the amount of the control amplicon, the latter representing the total amount of NOTCH1 DNA irrespective of its mutational status. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4×44K Agilent platform. The differential expression of specific genes/proteins was validated by QRT-PCR, western blotting and immunohistochemistry. A BrdU uptake assay was used to evaluate proliferation of CLL cells by CpG/IL2 stimulation. Results: in a cohort of 380 IGHV-UM CLL, the c.7544–7545delCT NOTCH1 mutation was found in 83/380 (21.8%) cases. QRT-PCR revealed a percentage of NOTCH1 mutated DNA ranging from 1 to 37%. CLL cases carrying the c.7544–7545delCT NOTCH1 mutation (NOTCH1-Mut) showed higher NOTCH1 protein expression than CLL cases lacking NOTCH1-Mut employing monoclonal antibodies either recognizing the trans-membrane (mean fold increase=3) or the intra-citoplasmic (mean fold increase=2.1) NOTCH1 domain. A GEP comparing RNA from purified CLL samples of 5 NOTCH1-Mut CLL and 5 CLL lacking NOTCH1-Mut was performed, selecting the 5 NOTCH1-Mut cases among those with the higher percentages of NOTCH1 mutated DNA (percentages of NOTCH1 mutated DNA ranging from 15 to 37%). This approach selected the nucleophosmin 1 gene (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-Mut CLL cases. A higher expression of the above mentioned genes in NOTCH1-Mut CLL was validated in a wider series of 34 cases (18 NOTCH1-Mut cases; NPM1, p=0.03; RPS6, p=0.045; RPS10, p=0.048; RPS17, p=0.048; RPS28, p=0.049; RPSA, p=0.048; RPL7A, p=0.039; RPL18, p=0.041, respectively). Western blot analysis in 8 cases (4 NOTCH1-Mut cases) confirmed a higher NPM1 expression in NOTCH1-Mut cases (range of fold increase from 1.6 to 5.2) also at protein level. Consistently, lymph nodes preparations from NOTCH1-Mut CLL cases revealed a strong NPM1 staining both in nucleoli and cytoplasms. Finally, when stimulated in-vitro with the CpG/IL2 combination, NOTCH1-mut IGHV-UM CLL cells proliferated, as detected by a BrdU uptake assay (>10 fold increase over control), and up-regulated NPM1 both at transcript (mean fold increase=2.02 after 18 hours of CpG exposure, p=0.001) and protein (fold increase of 1.34 after 6 hours of CpG exposure) levels. Conclusion: NPM1 was identified as constitutively overexpressed in NOTCH1-Mut IGHV-UM CLL together with several ribosome-associated components. These findings are suggestive for an increased activity of the ribosomal machinery in NOTCH1-Mut IGHV-UM CLL as part of the molecular processes leading to control of CLL cell growth and survival in this clinically unfavourable disease subset. Disclosures: No relevant conflicts of interest to declare.
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Mandelboim, O., E. Bar-Haim, E. Vadai, M. Fridkin, and L. Eisenbach. "Identification of shared tumor-associated antigen peptides between two spontaneous lung carcinomas." Journal of Immunology 159, no. 12 (December 15, 1997): 6030–36. http://dx.doi.org/10.4049/jimmunol.159.12.6030.
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Abstract CTLs recognize antigenic peptides bound to MHC class I Ags on the cell surface of tumor cells. Tumor-associated Ag (TAA) peptides are 8 to 10 amino acids long and can be derived from normal, mutated, or viral proteins. The majority of T cell-defined Ags have been identified in human melanoma cells. These were shown to be commonly expressed by different allogeneic melanomas that share the same MHC molecule. We have recently isolated Kb-restricted TAA peptides, which are mutations of the gap junction protein connexin 37, from the spontaneous C57BL/6 Lewis lung carcinoma (3LL). These peptides, named MUT 1 and MUT 2, serve as CTL epitopes and can induce CTL activity in vivo. Using CTL cross-reaction assays, peptide extraction, HPLC fractionation, and reverse transcriptase-PCR amplification, we show that clones of another spontaneous C57BL/6 lung carcinoma, CMT 64, share TAA peptides with the 3LL carcinoma. Vaccination with synthetic MUT 1 or MUT 2 induces CTLs that efficiently lyse CMT 64-derived clones, protects mice from CMT 64 metastasis, and affords therapy of established CMT 64 metastases. Hence, shared CTL epitopes exist between two spontaneous murine lung carcinomas.
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Bai, Dan, Xudong Zhang, Yu Li, Jing Ni, and Kai Lan. "A Missense POU4F3 Variant Associated with Autosomal Dominant Midfrequency Hearing Loss Alters Subnuclear Localization and Transcriptional Capabilities." BioMed Research International 2021 (June 21, 2021): 1–6. http://dx.doi.org/10.1155/2021/5574136.
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Background. The pathogenic variant, POU class 4 transcription factor 3 (POU4F3), is reported to cause autosomal dominant nonsyndromic hearing loss (ADNSHL). Previously, we have examined a four-generation midfrequency sensorineural hearing loss (MFSNHL) family (no. 6126) and established POU4F3 c.602T>C (p.Leu201Pro) as a potential disease-causing variant. Objectives. We explored the structural and functional alterations that the c.602T>C (p.Leu201Pro) variant enforces on the POU4F3 protein. Methods. We utilized wild-type (WT) and mutant (MUT) POU4F3 c.602T>C plasmid incorporation into HeLa cells to assess functional changes, by immunofluorescence and luciferase assays. To predict protein structural alterations in the MUT versus WT POU4F3, we also generated 3D structures to compare both types of POU4F3 proteins. Results. The WT POU4F3 is ubiquitously present in the nucleus, whereas the MUT form of POU4F3 exhibits a more restricted nuclear presence. This finding is different from other publications, which report a cytoplasmic localization of the MUT POU4F3. We also demonstrated that, as opposed to WT POU4F3, the MUT POU4F3 had 40% reduced luciferase activity. Conclusions. The reduced nuclear presence, combined with reduced transcriptional activity, suggests that the POU4F3 c.602T>C variant alters cellular activity and may contribute to the pathogenicity of POU4F3-related hearing loss. It, also, provides more evidence of the pathophysiological characteristics of MFSNHL.
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Hsu, Tsung-Yuan, Ling-Nung Hsu, Shih-Yu Chen, and Bi-Tzen Juang. "MUT-7 Provides Molecular Insight into the Werner Syndrome Exonuclease." Cells 10, no. 12 (December 8, 2021): 3457. http://dx.doi.org/10.3390/cells10123457.
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Werner syndrome (WS) is a rare recessive genetic disease characterized by premature aging. Individuals with this disorder develop normally during childhood, but their physiological conditions exacerbate the aging process in late adolescence. WS is caused by mutation of the human WS gene (WRN), which encodes two main domains, a 3′-5′ exonuclease and a 3′-5′ helicase. Caenorhabditis elegans expresses human WRN orthologs as two different proteins: MUT-7, which has a 3′-5′ exonuclease domain, and C. elegans WRN-1 (CeWRN-1), which has only helicase domains. These unique proteins dynamically regulate olfactory memory in C. elegans, providing insight into the molecular roles of WRN domains in humans. In this review, we specifically focus on characterizing the function of MUT-7 in small interfering RNA (siRNA) synthesis in the cytoplasm and the roles of siRNA in directing nuclear CeWRN-1 loading onto a heterochromatin complex to induce negative feedback regulation. Further studies on the different contributions of the 3′-5′ exonuclease and helicase domains in the molecular mechanism will provide clues to the accelerated aging processes in WS.
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Grishin, D. V., D. D. Zhdanov, Ju A. Gladilina, V. S. Pokrovsky, O. V. Podobed, M. V. Pokrovskaya, S. S. Aleksandrova, A. L. Milyushkina, M. A. Vigovskiy, and N. N. Sokolov. "Construction and characterization of a recombinant mutant homolog of the CheW protein from Thermotoga petrophila RKU-1." Biomeditsinskaya Khimiya 64, no. 1 (January 2018): 53–60. http://dx.doi.org/10.18097/pbmc20186401053.
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In the work a recombinant chemotaxis protein CheW from Thermotoga petrophila RKU-1 (TpeCheW) and its mutant homolog (TpeCheW-mut) were created. It was shown that, despite the low homology with CheW prototypes from intestinal bacteria, these proteins didn't cause metabolic overload and were well expressed by cells of E. coli laboratory strains. We have discovered a broad spectrum of industrial valuable properties of the TpeCheW-mut protein such as stability in a wide range of temperatures and pH, high expression level, solubility and possibility of the application of a simple low-stage purification methodology with the use of preliminary heat treatment. Possible directions of the scientific and industrial application of this protein were claimed.
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Hering, Fromm, Bücker, Gorkiewicz, Zechner, Högenauer, Fromm, Schulzke, and Troeger. "Tilivalline- and Tilimycin-Independent Effects of Klebsiella oxytoca on Tight Junction-Mediated Intestinal Barrier Impairment." International Journal of Molecular Sciences 20, no. 22 (November 8, 2019): 5595. http://dx.doi.org/10.3390/ijms20225595.
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Abstract: Klebsiella oxytoca causes antibiotic-associated hemorrhagic colitis and diarrhea. This was attributed largely to its secreted cytotoxins tilivalline and tilimycin, inductors of epithelial apoptosis. To study whether Klebsiella oxytoca exerts further barrier effects, T84 monolayers were challenged with bacterial supernatants derived from tilivalline/tilimycin-producing AHC6 or its isogeneic tilivalline/tilimycin-deficient strain Mut-89. Both preparations decreased transepithelial resistance, enhanced fluorescein and FITC-dextran-4kDa permeabilities, and reduced expression of barrier-forming tight junction proteins claudin-5 and -8. Laser scanning microscopy indicated redistribution of both claudins off the tight junction region in T84 monolayers as well as in colon crypts of mice infected with AHC6 or Mut-89, indicating that these effects are tilivalline/tilimycin-independent. Furthermore, claudin-1 was affected, but only in a tilivalline/tilimycin-dependent manner. In conclusion, Klebsiella oxytoca induced intestinal barrier impairment by two mechanisms: the tilivalline/tilimycin-dependent one, acting by increasing cellular apoptosis and a tilivalline/tilimycin-independent one, acting by weakening the paracellular pathway through the tight junction proteins claudin-5 and -8.
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Zheng, Yi-Zheng, Xiao-Ru Ji, Yun-Yang Liu, Shuai Jiang, Xiang-Ying Yu, Zhi-Ping Jia, Yue Zhao, Jun-Qiao Zhang, Jia-Li Zhang, and Yi Kong. "WPK5, a Novel Kunitz-Type Peptide from the Leech Whitmania pigra Inhibiting Factor XIa, and Its Loop-Replaced Mutant to Improve Potency." Biomedicines 9, no. 12 (November 23, 2021): 1745. http://dx.doi.org/10.3390/biomedicines9121745.
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Kunitz-type proteins or peptides have been found in many blood-sucking animals, but the identity of them in leeches remained elusive. In the present study, five Kunitz-type peptides named WPK1-WPK5 were identified from the leech Whitmania pigra. Recombinant WPK1-WPK5 were expressed in Pichia pastoris GS115, and their inhibitory activity against Factor XIa (FXIa) was tested. WPK5 showed inhibitory activity against FXIa with an IC50 value of 978.20 nM. To improve its potency, the loop replacement strategy was used. The loop 1 (TGPCRSNLER) and loop 2 (QYGGC) in WPK5 were replaced by loop 1 (TGPCRAMISR) and loop 2 (FYGGC) in PN2KPI, respectively, and the resulting peptide named WPK5-Mut showed an IC50 value of 8.34 nM to FXIa, which is about 100-fold the potency of FXIa compared to that of WPK5. WPK5-Mut was further evaluated for its extensive bioactivity in vitro and in vivo. It dose-dependently prolonged APTT on both murine plasma and human plasma, and potently inhibited FeCl3-induced carotid artery thrombosis in mice at a dose of 1.5 mg/kg. Additionally, WPK5-Mut did not show significant bleeding risk at a dose of 6 mg/kg. Together, these results showed that WPK5-Mut is a promising candidate for the development of an antithrombotic drug.
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Gargini, Ricardo, Berta Segura-Collar, Beatriz Herránz, Vega García-Escudero, Andrés Romero-Bravo, Felipe J. Núñez, Daniel García-Pérez, et al. "The IDH-TAU-EGFR triad defines the neovascular landscape of diffuse gliomas." Science Translational Medicine 12, no. 527 (January 22, 2020): eaax1501. http://dx.doi.org/10.1126/scitranslmed.aax1501.
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Gliomas that express the mutated isoforms of isocitrate dehydrogenase 1/2 (IDH1/2) have better prognosis than wild-type (wt) IDH1/2 gliomas. However, how these mutant (mut) proteins affect the tumor microenvironment is still a pending question. Here, we describe that the transcription of microtubule-associated protein TAU (MAPT), a gene that has been classically associated with neurodegenerative diseases, is epigenetically controlled by the balance between wt and mut IDH1/2 in mouse and human gliomas. In IDH1/2 mut tumors, we found high expression of TAU that decreased with tumor progression. Furthermore, MAPT was almost absent from tumors with epidermal growth factor receptor (EGFR) mutations, whereas its trancription negatively correlated with overall survival in gliomas carrying wt or amplified (amp) EGFR. We demonstrated that the overexpression of TAU, through the stabilization of microtubules, impaired the mesenchymal/pericyte-like transformation of glioma cells by blocking EGFR, nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and the transcriptional coactivator with PDZ-binding motif (TAZ). Our data also showed that mut EGFR induced a constitutive activation of this pathway, which was no longer sensitive to TAU. By inhibiting the transdifferentiation capacity of EGFRamp/wt tumor cells, TAU protein inhibited angiogenesis and favored vascular normalization, decreasing glioma aggressiveness and increasing their sensitivity to chemotherapy.
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Marin, Miguel, Eloisa Jantus-Lewintre, Libia Sanz, Angel Garcia-Martin, Cristina Reinoso, Isabel Benet, David Montaner, et al. "Characterization of the Proteomic and Genomic Profiles of Chronic Lymphocytic Leukemia Patients with Distinct Clinical Prognosis According to the Mutational Status of the IgVH and BCL6 and Expression Level of CD38 and ZAP70." Blood 106, no. 11 (November 16, 2005): 3272. http://dx.doi.org/10.1182/blood.v106.11.3272.3272.
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Abstract Introduction: In recent years several molecular prognostic factors have been identified in Chronic Lymphocytic Leukemia B (B-CLL). These include mutations in the variable region of the immunoglobulin genes (IgVH), somatic mutations in the BCL6 gene (Leukemia (2004) 18, 743–746) and the expression level of CD38 and ZAP70. However its biological significance is not clear. In order to identify novel molecular markers with prognostic and therapeutic value we have analyzed the proteomic and genomic profile of 40 B-CLL patients (Binet stage A). Material and methods: 100 μg of total PBMC proteins were used for IEF followed by 2D electrophoresis. Image analysis of scanned gels was used to identify statistically significant differentially expressed proteins. Image acquisition and analysis were performed using the Image Master Platinum software. Selected spots were subjected to automatic digestion and the proteins were identified by MALDI-TOF (Voyager DE-Pro, Applied Biosystems) peptide mass fingerprint using the Protein Prospector software. To confirm the initial identification amino acid sequencing of selected peptide ions was carried out by collision-induced dissociation (CID) with a nESI-QTRAP mass spectrometer from Applied Biosystems. 100 ng of total RNA was used to analyze the expression profile by cDNA microarrays (Whole Human Genome U133 Plus 2.0 Array from Affymetrix). Differential gene expression has been analyzed using the corresponding module (pomelo) from the GEPAS web tools (http://www.gepas.org). Results and Discussion: We found 126 proteins and 25 genes exhibiting differential expression (p<0.05, Student t-test, FDR-adjusted for multiple testing contrasts). We found 34 proteins and 18 genes with different expression according to the mutational status of IgVH. We also classified patients according to mutations in the BCL6 gene, which we previously showed clinical relevance (Leukemia (2004) 18, 743–746), and found 36 proteins and 2 genes with differential expression. Next, we compared samples attending to the mutational status of both genes, results are summarized in the following table: Compared groups attending to the mutational status of IgVH and BCL6 genes targets IgVH wt/BCL6 wt IgVH mut/BCL6 mut 16 proteins / 10 genes IgVH mut/BCL6 wt 28 proteins / 9 genes Of particular interest is the comparision between BCL6 status in IgVH mutated patients where we found 27 proteins with differential expression but no difference in the gene expression profile. This result suggest that the bad prognostic associated to BCL6 mutation may be due solely to postransductional modification of proteins. CD38 and ZAP70 expression level have also been associated with bad clinical prognosis, however, we found no significant difference in the gene expression profile according to CD38 or ZAP70 expression level. We are currently assessing a wider number of patients in order to obtain stronger conclusions. The study of these proteins and genes may lead to a better understanding of the different clinical behaviour of these B-CLL forms.
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Jones, Richard Julian, Janine Arts, and Robert Z. Orlowski. "Inhibition of the Human Double Minute (HDM)-2 E3 Ubiquitin Ligase Activates Different Programmed Cell Death (PCD) Pathways in Models of Non-Hodgkin Lymphoma (NHL) with Wild Type (wt) and Mutant (mut) p53." Blood 112, no. 11 (November 16, 2008): 3618. http://dx.doi.org/10.1182/blood.v112.11.3618.3618.
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Abstract Background: The ubiquitin-proteasome pathway has been validated as a target for NHL with the recent approval of bortezomib for mantle cell lymphoma (MCL). In addition to anti-tumor activity, however, proteasome inhibitors have pleiotropic effects, including activation of anti-apoptotic heat shock proteins, and their use clinically is complicated by toxicities such as peripheral neuropathy. By targeting E3 ubiquitin ligases, which are involved in ubiquitination of only a small subset of cellular proteins, it may be possible to achieve more specific anti-tumor effects with a better therapeutic index. One attractive target is HDM-2, which is responsible for ubiquitination of the p53 tumor suppressor. Methods: To evaluate the therapeutic potential of agents targeting HDM-2, we studied the impact of the small molecule JNJ-26854165, an inhibitor of HDM-2-function, in both p53 wt and mut cell line models. Results: Treatment of wt p53 NHL cell lines with JNJ-26854165 induced a dose- and time-dependent inhibition of proliferation, with an IC50 in the 0.02–0.3 μM range. Cell death, which was typically seen within 48 hours of HDM-2 inhibition, occurred through induction of type I PCD, as judged by the appearance of increased staining with Annexin V and activation of caspase 3. While cell lines with mut p53 were generally less sensitive than their wt p53 counterparts, JNJ-26854165 remained potent, with an IC50 in the 0.05–0.6 μM range. The latter cell lines showed a longer kinetics of death, with PCD being seen within 72 hours of drug exposure. Notably, in these mut p53 cell lines, very little Annexin V staining or caspase 3 activation was seen, consistent with a minor role for type I PCD. Instead, mut p53 cell lines demonstrated an increased content of acidic vacuoles by acridine orange staining, increased expression of Beclin 1 and Sequestosome 1/p62, and conversion of microtubule-associated protein 1 light chain 3 form I to form II, consistent with activation of type II PCD, or autophagy. Also, electron microscopy showed an increased presence of autophagosomes and autolysosomes, further supporting the activation of this pathway. Combinations of JNJ-26854165 with other agents, including rapamycin, doxorubicin, and an inhibitor of Bcl 2, showed enhanced anti-proliferative effects in a sequence-dependent fashion, which were greatest when the chemotherapeutic preceded the HDM-2 inhibitor. Combination index analysis revealed that these interactions met criteria for synergy. Conclusions: Inhibition of the function of HDM-2 using JNJ-26854165 is a promising approach that is effective against both wt and mut p53 models by activating type I and type II PCD, respectively. The effectiveness of JNJ-26854165 was enhanced in combination with currently used chemotherapeutics in a sequence specific manner, providing a rationale for translation of this novel approach into the clinic.
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Shechter, Sharon, Sapir Ya’ar Bar, Hamdan Khattib, Matthew J. Gage, and Dorit Avni. "Riok1, A Novel Potential Target in MSI-High p53 Mutant Colorectal Cancer Cells." Molecules 28, no. 11 (May 31, 2023): 4452. http://dx.doi.org/10.3390/molecules28114452.
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The vulnerabilities of cancer cells constitute a promising strategy for drug therapeutics. This paper integrates proteomics, bioinformatics, and cell genotype together with in vitro cell proliferation assays to identify key biological processes and potential novel kinases that could account, at least in part, for the clinical differences observed in colorectal cancer (CRC) patients. This study started by focusing on CRC cell lines stratified by their microsatellite (MS) state and p53 genotype. It shows that cell-cycle checkpoint, metabolism of proteins and RNA, signal transduction, and WNT signaling processes are significantly more active in MSI-High p53-WT cell lines. Conversely, MSI-High cell lines with a mutant (Mut) p53 gene showed hyperactivation of cell signaling, DNA repair, and immune-system processes. Several kinases were linked to these phenotypes, from which RIOK1 was selected for additional exploration. We also included the KRAS genotype in our analysis. Our results showed that RIOK1’s inhibition in CRC MSI-High cell lines was dependent on both the p53 and KRAS genotypes. Explicitly, Nintedanib showed relatively low cytotoxicity in MSI-High with both mutant p53 and KRAS (HCT-15) but no inhibition in p53 and KRAS WT (SW48) MSI-High cells. This trend was flipped in CRC MSI-High bearing opposite p53-KRAS genotypes (e.g., p53-Mut KRAS-WT or p53-WT KRAS-Mut), where observed cytotoxicity was more extensive compared to the p53-KRAS WT-WT or Mut-Mut cells, with HCT 116 (KRAS-Mut and p53-WT) being the most sensitive to RIOK1 inhibition. These results highlight the potential of our in silico computational approach to identify novel kinases in CRC sub-MSI-High populations as well as the importance of clinical genomics in determining drug potency.
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Pozzo, Federico, Tamara Bittolo, Erika Tissino, Francesca Maria Rossi, Riccardo Bomben, Antonella Zucchetto, Dania Benedetti, et al. "NOTCH1 Mutated IGHV Unmutated Chronic Lymphocytic Leukemia Cells Are Characterized By a Constitutive Overexpression of Nucleophosmin-1 and Ribosome-Associated Components." Blood 124, no. 21 (December 6, 2014): 3308. http://dx.doi.org/10.1182/blood.v124.21.3308.3308.
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Abstract Background: stabilizing mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM), immuno-chemorefractory or advanced disease phase CLL, and have been associated with particularly unfavourable prognosis (Rossi et al, Blood, 2012; Del Poeta et al, Br J Haematol, 2013; Stingelbauer et al, Blood, 2014). In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain (about 80% of which are a 7544-7545delCT frameshift deletion) and cause an accumulation of an active NOTCH1 isoform. Aim: to identify molecular/biological features of NOTCH1 mutated CLL. Methods: the presence of NOTCH1 mutations was investigated by ARMS-PCR or by direct sequencing. The percentage of NOTCH1 mutated DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR) and/or by next-generation-sequencing. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4x44K platform. Specific gene/protein validations were performed by QRT-PCR, western blotting, flow cytometry and immunofluorescence. Cell proliferation was evaluated by Cell Trace Violet assay. CLL-like cell line MEC-1 was transfected with a vector either with a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or with a NICD carrying a missense mutation (c.5304G>A), generating a stop codon at the beginning of the sequence, as control. Results:i) in a cohort of 588 IGHV-UM CLL, 144/588 (24.5%) cases carried NOTCH1 mutations (NOTCH1-mut cases), with a percentage of NOTCH1 mutated DNA ranging from 1% to 37%. NOTCH1-mut cases (8 cases; 11%-37% of NOTCH1 mutated DNA) showed higher NOTCH1 protein expression than NOTCH1 wild type cases (NOTCH1-wt, 11 cases) employing monoclonal antibodies either recognizing the trans-membrane (mean fold-change=3.0) or the intracellular (mean fold-change=2.1) NOTCH1 domain. ii) A GEP comparing purified cells of 5 IGHV-UM NOTCH1-mut cases (15%-37% of NOTCH1 mutated DNA) and 5 IGHV-UM NOTCH1-wt cases selected nucleophosmin-1 (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-mut CLL. A higher expression of NPM1 and of the genes codifying for 4 ribosomal proteins in NOTCH1-mut cases was validated in a wider independent series of 34 cases by QRT-PCR (18 NOTCH1-mut cases).iii) Western blot in 19 cases (8 NOTCH1-mut cases) confirmed a higher NPM1 protein expression in NOTCH1-mut cases (1.3-5.2 range of fold-change) also at protein level. When intracellular distribution and fluorescent intensity of NPM1 immunostaining were evaluated, an additional cytoplasmic staining was visible in NOTCH1-mut cases, along with a clear nucleolar staining visible in both NOTCH1-mut and NOTCH1-wt cases. NPM1 protein expression was also determined by an intra-nuclear staining by flow cytometry. By using this approach, 2 NOTCH1-mut cases were sorted according to NPM1 expression; notably, the NPM1high subpopulation showed a higher NOTCH1 mutational load than the NPM1low subpopulation. iv) MEC-1 cells transfected with mutated NICD showed higher NOTCH1 transcript and protein levels than MEC-1 cells transfected with the control vector (increments over the control: NICD transcript =2.2, NICD protein =1.3). These NICD mutated MEC-1 cells were characterized by higher NPM1 transcript and protein levels than the MEC-1 cells transfected with the control vector (increments over the control: NPM1 transcript =2.0, NPM1 protein =1.5). By performing a cell-trace-violet proliferation assay, NICD mutated MEC-1 cells were characterized by higher proliferation rates than MEC-1 cells transfected with the control vector (p=0.018). Finally, when the transfected MEC-1 cells were treated with 0.5 microM etoposide for 48 hours, NICD mutated MEC-1 cells presented higher viability rates than the MEC-1 cells transfected with the control vector, as evaluated by Annexin V/7-AAD staining (percentage of viable cells: 70% vs. 50%). Conclusions: NPM1 was constitutively overexpressed in NOTCH1-mut IGHV-UM CLL together with several ribosome-associated components. An increased activity of the ribosomal machinery/DNA-repair mechanisms (Lindstrom MS. Biochem.Res.Int. 2011) in NOTCH1-mut CLL can confer proliferative advantages and resistance to chemotherapeutic agents. Disclosures No relevant conflicts of interest to declare.
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Минакина, Liliya Minakina, Непомнящих, Svetlana Nepomnyashchikh, Егорова, Irina Egorova, Гуцол, Lyudmila Gutsol, Ясько, and Mikhail Yasko. "MISMATCH REPAIR AND REPAIR OF INSERTION/DELETION LOOPS IN EUKARYOTIC DNA." Бюллетень Восточно-Сибирского научного центра Сибирского отделения Российской академии медицинских наук 1, no. 3 (June 20, 2016): 72–75. http://dx.doi.org/10.12737/21614.
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The mismatch repair (MMR) system detects non-Watson – Crick base pairs as well as the defects, appearing in course of DNA replication, and helps to eliminate them by catalyzing the excision of the defect-containing region of daughter DNA and its error-free resynthesis. Thus, MMR remarkably improves the fidelity of replication. After separation, both strands contain non-repairable damages and the mismatches may generate DNA mutation in 50 % of cell progeny after next replication. MMR dysfunction causes surge of mutation rate, abnormal recombination, and cancer in humans and animals. Therefore, the main MMR efficiency parameter is mismatch correction before the next replication cycle. Mismatch detection is made by the MSH2 protein, which forms a heterodimer with either MSH6 or MSH3 (Mut S), depending on the damage (MSH6 is needed for the amendment of single base mispairs, whereas both MSH3 and MSH6 can correct IDLs). A heterodimer of MLH1 and PMS2 (Mut L) controls the interaction between the mismatch-detecting complex of proteins and other proteins essential for MMR, including exonuclease 1, helicase, nuclear antigen of proliferating cells, single-stranded DNA-binding protein and DNA polymerases δ and ε. MLH1 can form a heterodimer with two additional proteins – MLH3 and PMS1. PMS2 is required for the correction of single based mismatches, and PMS2 and MLH3 contribute to the correction of IDLs. The Nobel Prize in Chemistry 2015 was awarded for the studies of DNA repair, i.a. MMR.
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Grishin, D. V., Ju A. Gladilina, D. D. Zhdanov, M. V. Pokrovskaya, I. Yu Toropygin, S. S. Aleksandrova, V. S. Pokrovskiy, and N. N. Sokolov. "Preparation and characterization of a new mutant homolog of chemotaxis protein CheY from anaerobic hyperthermophilic microorganism Thermotoga naphthophila." Biomeditsinskaya Khimiya 65, no. 1 (January 2019): 41–50. http://dx.doi.org/10.18097/pbmc20196501041.
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Using genetic engineering methods the expression vectors structures have been designed to produce recombinant proteins TnaCheY and Tna CheY-mut, the homologues of the chemotaxis protein CheY from the hyperthermophilic organism Thermotoga naphthophila in Escherichia coli BL21(DE3) cells. The cultivation conditions of transformed strains were optimized. The influence of episomal expression of the heterologous chemotaxis protein CheY on growth kinetics parameters of the culture of mesophilic bacteria E. coli was studied. The optimal purification flowchart of the obtained proteins using thermolysis is proposed. Using the E. coli BL21(DE3) laboratory strain as an example, the possibility of employment the episomal expression of such proteins to control the cultivation and production time of pharmaceutically and industrially valuable metabolites due to the impact on some stages of the bacterial chemotaxis is experimentally proved.
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Logothetis, Constantine Nick, Chetasi Talati, Gregoire Calon, Nathan P. Horvat, Virginia Olivia Volpe, Nicole D. Vincelette, Andrew T. Kuykendall, et al. "Outcomes of IDH1/2 mutant AML patients treated with IDH1/2 inhibitors: A single institutional experience." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e19010-e19010. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e19010.
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e19010 Background: Recent studies showed that IDH1/2 are frequently mutated in AML and that aberrant 2-HG elevation driven by the mutant IDH1/2 proteins plays a pivotal role in AML development. Subsequent clinical trials of IDH1/2 inhibitors demonstrated promising outcomes in IDH1/2mut AML patients. In this single institutional retrospective study, we explored the efficacy and safety outcomes of IDH1/2mut AML patients treated with Ivosidenib or Enasidenib. Methods: We retrospectively identified AML patients who had IDH1/2 somatic mutations based on NGS assessments. Clinical and demographic data were extracted from the medical records. Statistical analyses were performed using GraphPad Prism (v.7.03) and SPSS (v.24.0). Results: A total of 43 ( IDH1mut, n = 12; IDH2mut, n = 33; both IDH1/2mut, n = 2) patients were included in the study. Median age at AML diagnosis was 67.6 (24.2-83.3) years and 24 (55.8%) patients were male. Eighteen (42%) patients had secondary AML and 13 (34.2%), 17 (44.7%), and 8 (21.1%) patients had favorable, intermediate, and adverse risk, respectively. A total of 23 (53.5%) and 9 (20.9%) patients received intensive chemotherapy and hypomethylating agents as their 1st line therapy. One patient received Enasidenib as the 1st line therapy and the rest of the patients had relapsed/refractory disease prior to IDH1/2 inhibitor therapy. Median number of treatment prior to IDH1/2 inhibitors was 4 (0-8). The median duration of IDH1/2 inhibitor treatment was 3.2 (0.2-31.6) months ( IDH1 mut, 2.5 [0.7-13.5]; IDH2 mut, 3.4 [0.2-31.6]). Treatment response was assessed in 38 patients and 18 had overall response (CR, n = 7 [18.4%]; PR, n = 11 [28.9%]). Among these, 13 patients had concurrent somatic mutations in FLT3, KRAS, NRAS, or PTPN11. The overall response rate in these patients was not statistically different compared to patients who did not have these mutations (38.5% vs. 40%, p > 0.05). The median PFS was 3.9 (0.4-14.7) months ( IDH1 mut, 5.6 [1.7-11.5] vs. IDH2 mut, 3.7 [0.4-14.7], p > 0.05) and median OS was 7.6 (0.4-44.1) months. The most common reason for IDH1/2 inhibitor discontinuation was disease progression (n = 21) followed by adverse events (n = 3) and allogeneic transplant (n = 2). The adverse events were assessed in 41 patients and the most common adverse events were differentiation syndrome ( IDH1 mut, n = 3; IDH2 mut, n = 5) and leukocytosis ( IDH1 mut, n = 4; IDH2 mut, n = 4) followed by hepatic toxicity ( IDH2 mut n = 7), and QTc prolongation ( IDH1 mut, n = 3). Conclusions: Our study indicates that IDH1/2 inhibitors remain a reasonable option for the refractory/relapsed IDH1/2mut AML. However, significant number of patients failed to show any response and many of the patients who showed initial response had short response duration. These findings warrant further studies to identify underlying resistance mechanisms of IDH1/2 inhibitors and the optimal combination therapeutic strategies.
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Garon, Edward B., Teresa Moran, Fabrice Barlesi, Leena Gandhi, Lecia V. Sequist, Sang-We Kim, Harry J. M. Groen, et al. "Phase II study of the HSP90 inhibitor AUY922 in patients with previously treated, advanced non-small cell lung cancer (NSCLC)." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): 7543. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.7543.
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7543 Background: AUY922 is a highly potent, non-geldanamycin, HSP90 inhibitor. A Phase I study in advanced solid tumors provided a recommended Phase II dose of 70 mg/m2. HSP90 acts as a chaperone of client proteins, including those relevant in NSCLC pathogenesis. This Phase II study assessed AUY922 in pts with previously treated advanced NSCLC stratified by molecular status. Methods: Pts with advanced NSCLC who progressed following ≥2 prior lines of chemotherapy, received AUY922 70 mg/m2 as a 1-hr infusion once-weekly. Pts were assigned to 4 strata: EGFR activating mutations (EGFR mut), KRAS mut, ALK rearranged (ALK+), or EGFR/KRAS/ALK wild type (wt). Phase II Bayesian hypothesis testing design allowed sharing of information between strata based on assessing similarity in observed response data. Primary endpoint was confirmed (RECIST) objective response rate (ORR) or stable disease (SD) at 18 wks. Secondary endpoints included OS, PFS, and safety/tolerability. Results: A total of 112 pts (median age 60 years; 45% male; 86% adenocarcinoma; 28 [25%] pts KRAS mut; 35 [31%] EGFR mut; 14 [12%] ALK+; 31 [28%] EGFR/KRAS/ALK wt) were treated at the December 16th 2011 cutoff. Most pts were heavily pretreated: 61% had received ≥3 prior systemic regimens; 64% had WHO PS 1 or 2. Mean duration of exposure was 9.1 wks. The most frequent adverse events (AEs, any grade [Gr]) were diarrhea (73%), visual AEs (71%), and nausea (43%). Most AEs were Gr 1/2; Gr 3/4 AE’s were rare (all <10%). The study is ongoing, and 29 pts were still receiving treatment at the cutoff date. Preliminary clinical activity of AUY922 (RECIST; investigator assessed) was seen with partial responses in 13/101 (13%) pts: 2/8 (25%) ALK+ pts, 6/33 (18%) EGFR mut pts, 4/30 (13%) EGFR/KRAS/ALK wt pts, 0/26 (0%) KRAS mut pts, and 1/4 (25%) pts of unknown status. In ALK+ pts, responses were seen in crizotinib (CRZ)-naive pts, and SD was seen with tumor shrinkage in CRZ-resistant pts. PFS data will be presented. Conclusions: AUY922 had an acceptable safety profile and demonstrated preliminary activity in heavily pretreated pts with advanced NSCLC. This trial demonstrates clinical activity of AUY922 in EGFR mut and EGFR/KRAS/ALK wt NSCLC pts in addition to ALK+ pts.
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Raychaudhury, Paromita, and Kenneth J. Marians. "The recombination mediator proteins RecFOR maintain RecA* levels for maximal DNA polymerase V Mut activity." Journal of Biological Chemistry 294, no. 3 (November 27, 2018): 852–60. http://dx.doi.org/10.1074/jbc.ra118.005726.
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Rumyantsev, Andrey M., Mikhail A. Tsygankov, Vladislav V. Veretennikov, Elena V. Sambuk, and Marina V. Padkina. "Heterologous synthesis of N and M fragments of capsid protein VP2 of avian infectious bursal disease virus in yeast Pichia pastoris." Ecological genetics 20, no. 1 (May 20, 2022): 49–59. http://dx.doi.org/10.17816/ecogen83441.
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BACKGROUND: Infectious bursal disease is one of the most common and economically important viral diseases of birds. Vaccination is currently the most effective way to control IBD. Subunit vaccines contain only the immunogenic protein of the pathogen or its fragments, but do not contain other proteins, lipopolysaccharides, toxins, which avoids vaccination side effects.
AIM: The aim of the work was to obtain yeast Pichia pastoris strains that synthesize and secrete the fragments of major coat protein VP2 of the infectious bursal disease virus.
MATERIALS AND METHODS: The DNA sequences encoding the N and M fragments of VP2 protein, were cloned under the control of the AOX1 gene promoter and integrated into the genome of P. pastoris strains X-33 (mut+) and GS115 (his4).
RESULTS: The analysis of proteins secreted by the obtained strains revealed the presence of additional proteins with a molecular weights corresponding to the target proteins.
CONCLUSIONS: Thus, the obtained strains of P. pastoris producers of N and M fragments of VP2 protein can be used for antigen production to create a subunit vaccine against avian IBD.
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Matic, I., M. Radman, and C. Rayssiguier. "Structure of recombinants from conjugational crosses between Escherichia coli donor and mismatch-repair deficient Salmonella typhimurium recipients." Genetics 136, no. 1 (January 1, 1994): 17–26. http://dx.doi.org/10.1093/genetics/136.1.17.
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Abstract To get more insight into the control of homologous recombination between diverged DNA by the Mut proteins of the long-patch mismatch repair system, we have studied interspecies Escherichia coli/Salmonella typhimurium recombination. Knowing that the same recombination pathway (RecABCD) is responsible for intraspecies and interspecies recombination, we have now studied the structure (replacement vs. addition-type or other rearrangement-type recombinants) of 81 interspecies recombinants obtained in conjugational crosses between E. coli donor and mutL, mutS, mutH, mutU or mut+ S. typhimurium recipients. Taking advantage of high interspecies sequence divergence, a physical analysis was performed on one third of the E. coli Hfr genome, which was expected to be transferred to S. typhimurium F- recipients during 40 min before interruption of the mating. Probes specific for each species were hybridized on dot blots of genomic DNA, or on colonies, and the composition of the rrn operons was determined from purified genomic DNA. With very few exceptions, the structure of these interspecies recombinants corresponds to replacements of one continuous block of the recipient genome by the corresponding region of the donor genome.
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Ntoufa, Stavroula, Stamatia Laidou, Fotis Psomopoulos, Marina Gerousi, Larry Mansouri, Georgios Tsiolas, Nikos Papakonstantinou, et al. "RPS15 mutations Repress mRNA Translation in Chronic Lymphocytic Leukemia Cells." Blood 132, Supplement 1 (November 29, 2018): 1843. http://dx.doi.org/10.1182/blood-2018-99-118150.
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Abstract We and others recently reported mutations within the RPS15 gene, encoding a component of the 40S ribosomal subunit, in clinically aggressive chronic lymphocytic leukemia (CLL). RPS15 mutations resided within an evolutionary conserved region, alluding to an oncogenic rather than a tumor-suppressor role. Our pilot functional analysis revealed that, similar to other ribosomal proteins (RPs), RPS15 also binds MDM2 and may impact on the p53 response. Here, we performed ribosome profiling in order to gain global insight into changes in translation induced by RPS15 mutations in CLL cells. This technique involves measuring translational efficiency (TE), by comparing the levels of ribosome-associated mRNA footprints against the total mRNA for each gene. For 6 CLL cases bearing mutant (mut, n=3) or wildtype (wt, n=3) RPS15, we obtained both ribosome-protected footprints (RPFs) and matching mRNA sequencing data. In parallel, we created stable MEC1 CLL cell lines expressing an additional copy of wt or mut RPS15 (131S) by lentiviral transduction; validation of the transgene expression was performed by Sanger sequencing of amplified cDNAs. Ribosome footprinting and subsequent library preparation of RPFs and total mRNA for all samples was performed with the Illumina Truseq Ribo Profile Kit and all libraries were sequenced on a NextSeq500 instrument. Reads were aligned to the human hg19 genome using Bowtie2. SystemPipeR was used to determine the percentage of reads mapping to 5' UTRs, CDS, and 3' UTRs and triplet periodicity was assessed using RibORF. The RPFs were of high quality, as assessed by expected RPF size (28-30nt), CDS enrichment, and triplet periodicity. To determine differentially expressed genes between RPS15-mut vs RPS15-wt cases we used DESeq2 while, for differentially translated genes we used Xtail. Changes in transcription and translation between PRS15-wt vs RPS15-mut cases showed limited overlap in both primary CLL cells and cell lines (12.8% and 12.9%, respectively), indicating the potential of ribosome profiling to reveal additional information compared to RNA sequencing alone. In primary CLL cells, 474 genes showed differences only at the transcription level (log2FC mRNA>I1I, p<0.05), while 742 genes were modulated only at the translation level (log2FC RPF>I1I, p<0.05). We identified 322 genes with differential TE (log2FC TE<I1I, p<0.05) between PRS15-wt vs RPS15-mut CLL cases; 262/322 (81%) showed reduced TE in RPS15-mut versus RPS15-wt cases. Similar analysis for the stable MEC1 cell lines revealed 749 genes displaying differences only at the transcription level (log2FC mRNA>I1I), while 1859 genes were regulated only at the translation level (log2FC RPF>I1I). Overall, 771 genes displayed differential TE (log2FC TE<I1I, adjusted p<0.1) between PRS15-wt vs RPS15-mut MEC1 cell lines; 48% of the genes showed reduced TE in mut vs wt cell lines and the remaining 52% augmented TE. The slightly different results compared to those obtained from primary CLL cells, may be attributed to the following reasons: (i) MEC1 cells are TP53-aberrant; (ii) the PRS15-wt cell line overexpresses the RPS15 gene compared to primary CLL cells; and,( iii) the RPS15-mut cell line expresses both the wt and mut RPS15 mRNAs (22% of the mapped reads correspond to the mut RPS15 and 78% to the wt gene, respectively). Gene ontology analysis (Enrichr) of the genes showing differential TE, revealed that in both primary CLL cells and MEC1 cell lines a large fraction of the deregulated transcripts is implicated in RNA binding processes (adj-p=0.0001; adj-p=1.98X10^-13, respectively) which are known to induce translational repression. Interestingly, in primary CLL cells, amongst genes with reduced TE we identified genes implicated in tRNA biosynthesis, protein processing in the endoplasmatic reticulum and the Hippo signaling pathway (p<0.01). Additionally, enrichment analysis revealed that a proportion of genes with reduced TE were targets of the MYC transcription factor (adj-p=0.0005). RP genes, despite unchanged mRNA levels, showed changes in RPF levels and differential TE, suggesting that RPs are also deregulated at the translational level. In conclusion, we show that RPS15 mutations rewire the translation program of CLL cells by reducing the TE of critical molecules, including translation initiation factors and other regulatory elements. Disclosures Hadzidimitriou: Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding.
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Hoemann, C. D., N. Beaulieu, L. Girard, N. Rebai, and P. Jolicoeur. "Two Distinct Notch1 Mutant Alleles Are Involved in the Induction of T-Cell Leukemia in c-myc Transgenic Mice." Molecular and Cellular Biology 20, no. 11 (June 1, 2000): 3831–42. http://dx.doi.org/10.1128/mcb.20.11.3831-3842.2000.
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ABSTRACT We have previously characterized a large panel of provirus insertion Notch1 mutant alleles and their products arising in thymomas of MMTVD/myc transgenic mice. Here, we show that these Notch1 mutations represent two clearly distinct classes. In the first class (type I), proviral integrations were clustered just upstream of sequences encoding the transmembrane domain. Type I Notch1 alleles produced two types of mutantNotch1 RNA, one of which encoded the entire Notch1 cytoplasmic domain [N(IC)] and the other of which encoded a soluble ectodomain [N(EC)Mut] which, in contrast to the processed wild-type ectodomain [N(EC)WT], did not reside at the cell surface and became secreted in a temperature-dependent manner. A second, novel class of mutant Notch1 allele (type II) encoded a Notch1 receptor with the C-terminal PEST motif deleted (ΔCT). The type II Notch1ΔCT protein was expressed as a normally processed receptor [N(EC)WT and N(IC)ΔCT] at the cell surface, and its ectodomain was found to be shed into the extracellular medium in a temperature- and calcium-dependent manner. These data suggest that both type I and type II mutations generate two structurally distinct Notch1 N(EC) and N(IC) proteins that may participate in tumor formation, in collaboration with the c-myc oncogene, through distinct mechanisms. Constitutive type I N(IC) and type II N(IC)ΔCT expression may enhance Notch1 intracellular signaling, while secreted or shed type I N(EC)Mut and type II N(EC) proteins may differentially interact in an autocrine or paracrine fashion with ligands of Notch1 and affect their signaling.
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Berrino, Enrico, Maria Costanza Aquilano, Emanuele Valtorta, Vito Amodio, Giovanni Germano, Marco Gusmini, Ivana Sarotto, et al. "Abstract 5419: A unique pattern of heterogeneous MMR protein expression in colorectal cancer unveils different degrees of tumor mutational burden and distinct tumor microenvironment features." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5419. http://dx.doi.org/10.1158/1538-7445.am2022-5419.
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Abstract Introduction: Mismatch repair (MMR) protein expression in colorectal cancer (CRC) is usually homogeneously retained or lost. However, rare CRC lesions may show a heterogeneous pattern of MMR protein expression. We aimed to evaluate the molecular effect of a heterogeneous expression of the MMR proteins in a complex context of concomitant loss of one of the other effectors of the DNA repair mechanism. Experimental procedures: We evaluated MMR protein expression (by immunohistochemistry -IHC- for MLH1, MSH2, MSH6 and PMS2) in 200 CRCs, identifying 3 groups with proficient (MMRp), deficient (MMRd) and heterogeneous (MMRh) MMR. MMRh tumors were micro-dissected based on the expression of the heterogeneous marker, DNA was extracted and subjected to targeted sequencing (TSO500 panel, Illumina). RNA was purified from bulk tumors of all MMRh cases and in a control series of 10 MMRp and 10 MMRd CRCs and analyzed using the IO360 panel (NanoString). The degree of tumor infiltrating lymphocytes (TILs) was calculated on H&E slides and on IHC sections stained with antibodies raised against CD8. Results: Twenty-nine out of 200 cases were MMRd (14.5%). Nine cases (4.5%) showed a heterogeneous pattern of MMR expression, with 6 tumors harboring concomitant loss of one of the other MMR proteins, resulting in a microsatellite instable (MSI) phenotype. Five out of the 9 cases were suitable for separate mutational analysis of IHC-positive and IHC-(double)negative components of the tumor. Both components of all the lesions exhibited a high tumor mutation burden (mean TMB: 59 mut/Mb) in line with the MSI status, nevertheless a significant increase in TMB in the double-negative components was observed (mean TMB: neg. 67 mut/Mb vs pos. 53 mut/Mb), due to a higher number of sub-clonal, non-synonymous variants compared to the positive component. Comparative gene expression analyses between MMRd, MMRp and MMRh CRCs highlighted differential gene expression patterns, with MMRh tumors displaying a strong activation of angiogenesis, MAPK and PI3K-AKT axes. Moreover, these tumors showed the highest number of TILs, characterized by a substantial population of exhausted CD8+ lymphocytes. Conclusions: We describe a unique subgroup of CRCs showing heterogeneous expression of MMR proteins, in a background of concomitant loss of one of the other markers. This heterogeneity is associated with i) an increase of TMB and of unstable loci in the negative component, with additional markedly sub-clonal variants, ii) a high number of TILs, mostly exhausted CD8+ lymphocytes, and iii) a differential activation of signaling pathways. Taken together these data provide indirect evidence that heterogeneous loss of MMR proteins may impact on the biology of CRCs. Whether this pattern may influence response to immune checkpoint inhibition remains to be determined. Citation Format: Enrico Berrino, Maria Costanza Aquilano, Emanuele Valtorta, Vito Amodio, Giovanni Germano, Marco Gusmini, Ivana Sarotto, Anna Sapino, Silvia Marsoni, Andrea Sartore-Bianchi, Alberto Bardelli, Salvatore Siena, Emanuela Bonoldi, Caterina Marchio. A unique pattern of heterogeneous MMR protein expression in colorectal cancer unveils different degrees of tumor mutational burden and distinct tumor microenvironment features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5419.
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Kornblau, Steven M., Chenyue W. Hu, Yihua Qiu, Suk Young Yoo, Trang T. Nguyen, Tapan Kadia, Gautam Borthakur, Kevin Coombes, and Amina A. Qutub. "Heatshock Protein (HSP) Family Expression Is Dichotomous and Prognostic in Acute Myelogenous Leukemia (AML)." Blood 124, no. 21 (December 6, 2014): 1026. http://dx.doi.org/10.1182/blood.v124.21.1026.1026.
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Abstract Background. Heat shock family proteins (HSP) are induced by cellular stress, and act as molecular chaperones regulating protein folding, conformation and proteosomal degradation, thereby affecting cell stability, cycling, and apoptosis. High levels of some HSP have been observed in AML and high HSP90 is prognostically adverse. Cell line studies show that HSP protects AML cells against chemotherapy or other stresses and that inhibition of HSP 27, 70 or 90 has antileukemic activity. HSP proteins have typically been studied individually, and in small series. Interactions with multiple signaling and functional pathways are suspected, but the actual relationship in primary AML samples is unknown. We therefore investigated the protein expression of members of the HSPA, B and C groups, HSP 70, 27 and 90, Hugo Names HSPA1A_L, HSPB1 and HSP90AA1_B1, respectively, in a large series of 511 AML patients and compared expression to 228 other simultaneously measured proteins. Methods. We made a reverse phase protein array (RPPA) with protein from leukemia enriched cells from 511 new AML patients. Both bone marrow (BM, n=387) and peripheral blood (PB, n=283) samples were used, with 140 cases having both. The RPPA was probed with 231 strictly validated antibodies, including antibodies against HSP27, 70 and 90. Expression was compared to that of normal bone marrow derived CD34+ cells. Interaction networks with the other 228 proteins were generated from the RPPA data using glasso and supplemented by the literature of known interactions. Results: A heatmap of HSP27, 70 and 90 expression was generated and hierarchical k-and means clustering performed (Fig 1A). Using the Prototype Clustering method an optimal division into two clusters C1) characterized by High HSP27 and 70 but low HSP90 and C2) characterized by High HSP90 and low HSP27 and 70 expression. Patients with high HSP90 had significantly higher % BM Blasts (63.6% vs. 42%), WBC (40.8K vs 16.9K), %PB blasts (44 vs. 17%), LDH levels, concurrent infection 34% vs. 12%), FLT3-ITD (25 vs. 13%) or FLT3-D835 mutation (8% vs. 4%) (all P < 0.00001) compared to those with High HSP27 & 70. HSP90 High patients were less likely to have an antecedent hematological disorder (26 vs. 47%, P < 0.00001). Other clinical features, age, gender, cytogenetics, RAS mutation, NPM1 mutation did not differ. Overall survival for all patients (Fig D) was inferior for those with High HSP90 (42 vs. 60 weeks, p = 0.04), but this was based solely on those with intermediate cytogenetics (Fig E) (48 vs. 91 weeks p =< 0.00001), whether FLT-3 mutant (Fig G) (30 vs. 78 weeks, p=0.025) or Wildtype (Fig H, p = 0.037). This was seen in NPM1-WT/FLT3- WT (p=0.03), NPM1-Mut/FLT3 -Mut (fig I, p=0.006) and NPM1-Mut/FLT3 WT (fig J, p=0.08). High HSP90 patients also had inferior remission duration (Fig F, 39 vs. 84 weeks, p=0.025). Networks (Fig B & C) of proteins with expression strongly (p <.00001) correlated with the HSP in the literature (dashed spoke) or the dataset (solid spoke) are shown. Differential expression between the two groups are shown in Fig C (*). Among those with differential expression, levels were higher in the HSP90 high group for histone methylation associated proteins ASH2L, SIRT1, hnRNPK, Nucleolin, proliferation regulators Myc, EIF2S1, EIF2AK2, EIF4E, and for apoptosis/autophagy proteins Beclin1, Park7, MDM2 and BCL2. In contrast protein levels were lower in HSP90 High for : AKT1.pT308, BIRC2, CDKN1A, CDKN2A, CDK4, EGFR 7 pEGFR, ERBB2, HIF1α, IGF1R, LCK PKCα, SRC & pSRC. Conclusions. AML is characterized by dichotomous expression of HSP proteins 27, 70 and 90. HSP expression pattern is associated with FLT3 mutation but not cytogenetics. HSP90 expression is prognostically adverse, exclusively among those with intermediate cytogenetics. High HSP90 expression is associated with higher WBC and PB and BM blast percentages, suggesting a proliferative advantage. This may arise through the effects of Myc and through increased gene expression arising from increased histone3K4 trimethylation from ASH2L and decreased histone 3 acetylation due to increased Sirt1 levels. Therapeutics directed at interfering with HSP function will need to consider which HSP pattern a patient exhibits to target the correct family members. Figure 1 Figure 1. Disclosures Borthakur: Tetralogic Pharmaceuticals: Research Funding.
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Yang, Hyeon, Bo Ram Lee, Hwi-Cheul Lee, Sun Keun Jung, Ji-Youn Kim, Jingu No, Sureshkumar Shanmugam, et al. "Isolation and characterization of cultured chicken oviduct epithelial cells and in vitro validation of constructed ovalbumin promoter in these cells." Animal Bioscience 34, no. 8 (August 1, 2021): 1321–30. http://dx.doi.org/10.5713/ab.20.0627.
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Objective: Transgenic hens hold a great promise to produce various valuable proteins. Through virus transduction into stage X embryo, the transgene expression under the control of constructed chicken ovalbumin promoters has been successfully achieved. However, a validation system that can evaluate differently developed ovalbumin promoters in <i>in vitro</i>, remains to be developed. Methods: In the present study, chicken oviduct epithelial cells (cOECs) were isolated from oviduct tissue and shortly cultured with keratinocyte complete medium supplemented with chicken serum. The isolated cells were characterized with immunofluorescence, western blot, and flow cytometry using oviduct-specific marker. Chicken mutated ovalbumin promoter (Mut-4.4-kb-pOV) was validated in these cells using luciferase reporter analysis. Results: The isolated cOECs revealed that the oviduct-specific marker, ovalbumin protein, was clearly detected by immunofluorescence, western blot, and flow cytometry analysis revealed that approximately 79.40% of the cells contained this protein. Also, luciferase reporter analysis showed that the constructed Mut-4.4-kb-pOV exhibited 7.1-fold (p<0.001) higher activity in the cOECs. Conclusion: Collectively, these results demonstrate the efficient isolation and characterization of cOECs and validate the activity of the constructed ovalbumin promoter in the cultured cOECs. The <i>in vitro</i> validation of the recombinant promoter activity in cOECs can facilitate the production of efficient transgenic chickens for potential use as bioreactors.
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Smith, Craig P., Elizabeth A. Potter, Robert A. Fenton, and Gavin S. Stewart. "Characterization of a human colonic cDNA encoding a structurally novel urea transporter, hUT-A6." American Journal of Physiology-Cell Physiology 287, no. 4 (October 2004): C1087—C1093. http://dx.doi.org/10.1152/ajpcell.00363.2003.
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Two closely related genes, UT-A ( Slc14a2) and UT-B ( Slc14a1), encode specialized transporter proteins that modulate the movement of urea across cell membranes. In this article, we report the characterization of a cDNA isolated from human colonic mucosa encoding a novel UT-A urea transporter, hUT-A6. The encoded protein is 235 amino acids (aa) in length, making it the smallest UT-A member characterized. On the basis of previous structural predictions, hUT-A6 is structurally unique in that it consists of a single hydrophobic core flanked by hydrophilic NH2- and COOH-terminal domains. The transcript encoding hUT-A6 contains a novel 129-bp exon, exon 5a, which, as a result of alternative splicing, introduces a unique 19-aa segment and a stop codon. Functionally, the protein transports urea, and this activity is inhibited by phloretin. Interestingly, despite the lack of a protein kinase A (PKA) consensus site {[RK]( 2 )-X-[ST]}, transport of urea by hUT-A6 is stimulated by PKA agonists. Deletion of the two PKA consensus sites from murine UT-A3 (mUT-A3) did not affect the stimulatory response of PKA agonists, which, together with the lack of PKA consensus sites in hUT-A6, indicates that regulation of hUT-A6 and mUT-A3 is not mediated through a classic PKA phosphorylation consensus.
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Wilkemeyer, M. F., E. R. Andrews, and F. D. Ledley. "Genomic structure of murine methylmalonyl-CoA mutase: evidence for genetic and epigenetic mechanisms determining enzyme activity." Biochemical Journal 296, no. 3 (December 15, 1993): 663–70. http://dx.doi.org/10.1042/bj2960663.
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Methylmalonyl-CoA mutase (MCM) is a nuclear-encoded mitochondrial matrix enzyme. We have reported characterization of murine MCM and cloning of a murine MCM cDNA and now describe the murine Mut locus, its promoter and evidence for tissue-specific variation in MCM mRNA, enzyme and holo-enzyme levels. The Mut locus spans 30 kb and contains 13 exons constituting a unique transcription unit. A B1 repeat element was found in the 3′ untranslated region (exon 13). The transcription initiation site was identified and upstream sequences were shown to direct expression of a reporter gene in cultured cells. The promoter contains sequence motifs characteristic of: (1) TATA-less housekeeping promoters; (2) enhancer elements purportedly involved in co-ordinating expression of nuclear-encoded mitochondrial proteins; and (3) regulatory elements including CCAAT boxes, cyclic AMP-response elements and potential AP-2-binding sites. Northern blots demonstrate a greater than 10-fold variation in steady-state mRNA levels, which correlate with tissue levels of enzyme activity. However, the ratio of holoenzyme to total enzyme varies among different tissues, and there is no correlation between steady-state mRNA levels and holoenzyme activity. These results suggest that, although there may be regulation of MCM activity at the level of mRNA, the significance of genetic regulation is unclear owning to the presence of epigenetic regulation of holoenzyme formation.
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Riegger, David, Rong Hai, Dominik Dornfeld, Benjamin Mänz, Victor Leyva-Grado, Maria T. Sánchez-Aparicio, Randy A. Albrecht, et al. "The Nucleoprotein of Newly Emerged H7N9 Influenza A Virus Harbors a Unique Motif Conferring Resistance to Antiviral Human MxA." Journal of Virology 89, no. 4 (December 10, 2014): 2241–52. http://dx.doi.org/10.1128/jvi.02406-14.
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ABSTRACTInterferon-induced Mx proteins show strong antiviral activity against influenza A viruses (IAVs). We recently demonstrated that the viral nucleoprotein (NP) determines resistance of seasonal and pandemic human influenza viruses to Mx, while avian isolates retain Mx sensitivity. We identified a surface-exposed cluster of amino acids in NP of pandemic A/BM/1/1918 (H1N1), comprising isoleucine-100, proline-283, and tyrosine-313, that is essential for reduced Mx sensitivity in cell culture andin vivo. This cluster has been maintained in all descendant seasonal strains, including A/PR/8/34 (PR/8). Accordingly, two substitutions in the NP of PR/8 [PR/8(mut)] to the Mx-sensitive amino acids (P283L and Y313F) led to attenuation in Mx1-positive mice. Serial lung passages of PR/8(mut) in Mx1 mice resulted in a single exchange of tyrosine to asparagine at position 52 in NP (in close proximity to the amino acid cluster at positions 100, 283, and 313), which partially compensates loss of Mx resistance in PR/8(mut). Intriguingly, the NP of the newly emerged avian-origin H7N9 virus also contains an asparagine at position 52 and shows reduced Mx sensitivity. N52Y substitution in NP results in increased sensitivity of the H7N9 virus to human Mx, indicating that this residue is a determinant of Mx resistance in mammals. Our data strengthen the hypothesis that the human Mx protein represents a potent barrier against zoonotic transmission of avian influenza viruses. However, the H7N9 viruses overcome this restriction by harboring an NP that is less sensitive to Mx-mediated host defense. This might contribute to zoonotic transmission of H7N9 and to the severe to fatal outcome of H7N9 infections in humans.IMPORTANCEThe natural host of influenza A viruses (IAVs) are aquatic birds. Occasionally, these viruses cross the species barrier, as in early 2013 when an avian H7N9 virus infected humans in China. Since then, multiple transmissions of H7N9 viruses to humans have occurred, leaving experts puzzled about molecular causes for such efficient crossing of the species barrier compared to other avian influenza viruses. Mx proteins are known restriction factors preventing influenza virus replication. Unfortunately, some viruses (e.g., human IAV) have developed some resistance, which is associated with specific amino acids in their nucleoproteins, the target of Mx function. Here, we demonstrate that the novel H7N9 bird IAV already carries a nucleoprotein that overcomes the inhibition of viral replication by human MxA. This is the first example of an avian IAV that is naturally less sensitive to Mx-mediated inhibition and might explain why H7N9 viruses transmitted efficiently to humans.
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Mooradian, Meghan, James M. Cleary, Justine Vanessa Cohen, Donald P. Lawrence, Elizabeth Iannotti Buchbinder, Anita Giobbie-Hurder, Aparna Raj Parikh, et al. "CTEP 9557: A dose-escalation trial of combination dabrafenib, trametinib, and AT13387 in patients with BRAF mutant solid tumors." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 3609. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.3609.
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3609 Background: Combination BRAF and MEK inhibitor therapy is associated with response in patients (pts) with BRAF mutant (mut) solid tumors; however critical limitations for the durable activity of these agents remains. Preclinically, the addition of heat shock protein 90 (HSP90) inhibitors improves the efficacy of BRAF inhibitor (BRAFi) therapy in both BRAFi -sensitive and resistant mutant cell lines. Methods: CTEP study 9557 (NCT02097225) is a phase I study designed to determine the safety and efficacy of the small molecule HSP90inhibitor, AT13387, in combination with dabrafenib (dab) and trametinib (tram) in patients with BRAF V600E/K mut solid tumors. Prior chemotherapy, immunotherapy, BRAF and/or MEK exposure was permitted. The primary objective was to determine the maximum tolerated dose (MTD). Results: From July 2015 to June 2018, 22 patients with previously treated, metastatic BRAF V600E/K mut solid tumors were enrolled using a 3 + 3 design at four dose levels (DL) (Table). Pts were predominantly female (59%) with a median age of 57.5yrs (37 -75). The most common tumor type was BRAF V600Emut colon cancer (N=12). Dose limiting toxicities (DLTs) occurred in one patient in DL3 and one in DL4, specifically grade 3 myelosuppression and fatigue, respectively. The MTD was Dab 150mg [BID/PO], Tram 2mg [QD/PO] and AT1187 260mg/m2 [D1,8,15/IV]. Twenty-one of 22 pts were eligible for efficacy assessment. Best response, per RECIST 1.1, was partial response (PR) in 2 pts – one with colon ca (TKI-naïve), one with melanoma (TKI-resistant) - stable disease (SD) in 8 pts, and disease progression (PD) in 11 with a disease control rate (PR + SD) of 47.6% (90% CI: 29% - 67%). Median time to progression was significantly longer in DL3 (3.9 mths; 1.8-9.2) compared to DL1 (1.6mths; 0.9-1.7) or DL2 (1.5; 0.6-3.6). Median PFS and OS were 1.8mths (90% CI: 1.6 – 3.7mths) and 5.1 mths (90% CI: 2.5 -10.6mths), respectively. Median OS was not reached in DL3/4. Correlative data on the expression of the key signaling proteins relating to response will be presented at the meeting. Conclusions: HSP90 inhibition combined with BRAF/MEK inhibition was determined to be safe with evidence of disease control in a heavily pre-treated population of pts with BRAF V600E/K mut solid tumors. Clinical trial information: NCT02097225 . [Table: see text]
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Cai, Xiuyu, Chunwei Xu, Wen xian Wang, Quxia Zhang, Yu Chen, Yong Fang, Hong Wang, et al. "Incidence of FGFR-TACC gene fusions in Chinese non-small cell lung cancer (NSCLC): A multicenter study." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e13001-e13001. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e13001.
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e13001 Background: Fibroblast growth factor receptors (FGFR) are transmembrane kinase proteins with growing importance in non-small cell lung cancer (NSCLC) biology given the frequency of molecular alterations and vast interface with multiple other signaling pathways. Furthermore, numerous FGFR inhibitors in clinical development demonstrate the expanding therapeutic relevance of this pathway. The aim of this study was to evaluate the prevalence of FGFR-TACC fusions in Chinese NSCLC populations, which had not been reported earlier, and to describe targeting potential in Chinese NSCLC populations. Methods: A multicenter study in China was initiated from February 2014, and NSCLC patients have been enrolled as of December 2018. Capture-based comprehensive genomic profiling was performed on 2743 NSCLC FFPE samples sequenced to a mean coverage depth of > 650X for up to 381 cancer-related genes. Genomic alterations (GA) included short variant (SV) base subs and insertions/deletions, copy number alterations, and rearrangements/fusions. Tumor mutational burden (TMB; mut/Mb) was calculated on up to 1.2 Mb of sequenced DNA. Results: Of this entire cohort, just 16 (0.58%) patients were identified with FGFR-TACC fusions, including FGFR1-TACC 1 fusion (1), FGFR2-TACC2 fusion (3) and FGFR3-TACC3 fusion (12). Median patient age was 57 (range 36-84 years). Of the FGFR-TACC fusion NSCLC patients, 56.25% were detected in female patients. Biopsies were obtained from primary lung tumor (31.25%) and metastatic sites (68.75%). Overall TMB in the FGFR-TACC fusion was low (median 3.6 mut/Mb), although two cases (12.50%) had > 20 mut/Mb. Of the FGFR-TACC fusion NSCLC patients, two cases (12.50%) featured EGFR SV alterations. Conclusions: FGFR-TACC fusions occur in a subset of patients with NSCLC. Such patients should be considered for clinical trials featuring FGFR inhibitors (AZD4547). Moreover, NGS can provide information for targeted therapy. For NSCLC patients to benefit from more personalized cancer treatment, clinical therapy should improve with clinical diagnostics through multi-gene assays to determine the actual clinical benefits.
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Wang, Chen, Yoshihiro Hayashi, Xiongwei Cai, Xiaomei Yan, Gang Huang, and Yi Zheng. "Inducible Correction of a RUNX1 Mutation of Human AML Causes a Switch of AML to B-ALL." Blood 132, Supplement 1 (November 29, 2018): 1325. http://dx.doi.org/10.1182/blood-2018-99-116919.
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Abstract Background: Point mutations of RUNX1 gene are frequently seen in myeloid malignancies such as myelodysplasia (MDS) and acute myeloid leukemia (AML), and are associated with unfavorable clinical outcomes. AML patients bearing RUNX1 mutations have a significantly lower complete remission (CR) rate compared with patients with wild-type RUNX1 (30% vs. 73%), whereas in contrast, the CR rate of ALL patients, in which RUNX1 is rarely mutated, are at 92%. It remains unclear whether RUNX1 mutations found in AML patients are causal to AML maintenance, and if RUNX1 mutations serve as a valid therapeutic target in the treatment of such therapy-resistant AML. Experimental Approach: The RUNX1 mutations are found frequently associated with MLL-partial tandem duplication (MLL-PTD) in AML. We have developed two complementary mouse models that express a MLL-PTDknock-in mutation together with a RUNX1 S291fsX300 mutation found in AML patients: in the first model RUNX1 S291fsX300 was introduced into the bone marrow cells of the MLL-PTD knock-in mice by retrovirus transduction, while in the second model the tetracycline-inducible RUNX1 S291fsX300 mutation was knock-in at the Collagen a1 locus and the mice was crossed to MLL-PTD knock-in. In the second model, the mutant RUNX1 protein is only expressed when the mice were fed with doxycycline containing food and the RUNX1 mutant is absent or "corrected" upon doxycycline withdrawn. BM cells of these mice, as well as WT and MLL-PTD controls, were transplanted to syngeneic mice and the recipients were tracked for disease development and progression at bi-monthly intervals. The doxycycline induction or withdraw was carried out after secondary transplant, and the expression of RUNX1 S291fsX300 was monitored by a built-in GFP reporter and verified by RT-PCR.Under tetracycline induction, the RUNX1 S291fsX300 and MLL-PTD double mutation bearing mice developed a spontaneously AML and had a survival time 6-10 months after transplantation, and they showed symptoms of MDS/AML or AML, including thrombocytopenia, anemia, leukocytosis, splenomegaly, abnormal BM and spleen cell morphologies. We transplanted the AML mouse bone marrow to secondary recipients, and observed them with or without continuing doxycycline induction. By using the O-propargyl-puromycin to track the newly synthesized proteins, we found that the RUNX1 mut-on and RUNX1 mut-off mice showed comparable protein synthesis rate at full-blown leukemia stage, indicating that the RUNX1 mutation does not play a restrictive role to protein synthesis in transformed leukemia cells. The c-Kit+ AML cells cultured from the RUNX1 mut-off mice showed reduced viability and significantly reduced CFU-C activities, which are accompanied by a lineage switch in a decrease in expression of Mac1 and an increase in expression of B220. In vivo, the RUNX1 mut-off mice showed a slightly prolonged survival compared with RUNX1 mut-on group (182 vs. 126 days, p=0.0036). An analysis of the lineage distribution in their peripheral blood and bone marrow revealed that the RUNX1 mut-off AML mice underwent a dramatic lineage switch from myelocytes to lymphocytes (Fig. 1): while the Mac1+Gr1+population remained high in the RUNX1 mut-on AML mice, turning off RUNX1 mut expression caused an increased B cell population in expense of the Mac1+Gr1+population. Pathological analysis also showed a drastic decrease of the myeloid marker MPO and increased lymphoid marker B220 in the RUNX1mu-off mice. Consistently, a RT-PCR test of the BM c-Kit+ cells further showed that upon turning off the RUNX1mut, critical B cell regulatory genes including TCF3, EBF1, PAX5, CD79A were significantly up regulated whereas myeloid regulatory genes such as PU.1 were downregulated. In addition, RUNX1 S291fsX300 expression in the AML cells confers a resistance to chemotherapy (Ara-C) treatment compared with that of WT RUNX1 expression, indicating that the RUNX1 mutation is responsible for the AML resistance to therapy. Conclusion:Although RUNX1 mutations found in AML patient may not be essential for the hyperproliferative and growth phenotype of the AML, they play a causal role in maintaining myeloid lineage restriction, expansion and chemo-resistance. Correction or removal of the RUNX1 mutation in AML cells leads to a disease switch from AML to B-ALL, effectively changing the difficult-to-treat drug-resistant AML to another disease with more favorable CR (B-ALL). Disclosures No relevant conflicts of interest to declare.
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Nurdiani, Dini, Hariyatun Hariyatun, and Wien Kusharyoto. "Secretory expression of human insulin precursor in Pichia pastoris employing truncated α-factor leader sequence and a short C-peptide." Indonesian Journal of Biotechnology 23, no. 2 (December 24, 2018): 102. http://dx.doi.org/10.22146/ijbiotech.38958.
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In the past ten years, diabetes prevalence has increased rapidly in low- and middle-income countries due to lifestyle changes. This increased number of diabetic patients leads to the escalation of recombinant insulin demand, which is creating a large global insulin market. Pichia pastoris has appeared as an alternative host to produce recombinant proteins. It has excellent qualifications as an expression host for large-scale production of recombinant proteins for therapeutic use. In this study, we attempted to express the insulin precursor (IP) in P. pastoris. We used a synthetic IP-encoding gene constructed in frame with the truncated α-factor secretory signal and a short C-peptide (DGK) linked A- and B-chain of human insulin in a pD902 expression vector. Several zeocin resistant clones were successfully obtained and verified with PCR using AOX1 specific primers for the integration of the expression cassette into the P. pastoris genome and for the identification of Mut phenotypes. The secretion of IP by the Pichia pastoris clone in the culture supernatant was confirmed using SDS-PAGE, where a single band of the secreted IP with a molecular mass above 6.5 kDa was found.
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Smoots, Stephen G., Anna R. Schreiber, Betelehem Yacob, Adrian Dominguez, Cecilia Levandowski, Todd M. Pitts, and Jennifer R. Diamond. "Abstract 136: Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines." Cancer Research 82, no. 12_Supplement (June 15, 2022): 136. http://dx.doi.org/10.1158/1538-7445.am2022-136.
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Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer which lacks HER2 overexpression, as well estrogen and progesterone receptor expression. TNBC constitutes 10-15% of all breast cancers but has a worse prognosis due to limited treatment options and high rates of metastatic recurrence. Doxorubicin (dox) remains one of the most active chemotherapy agents for the treatment of TNBC, although de novo and acquired resistance to chemotherapy remains a major challenge. The purpose of this work was to characterize dox resistant cell phenotypes and investigate treatment-induced senescence in p53-mutated (Mut) and wildtype (WT) TNBC cell lines. Methods: A panel of 12 TNBC cell lines (p53 WT and p53 Mut) were exposed to dox (0-5 uM) for 72 hours and cellular proliferation was determined using the Cell Titer Glo assay. A subset of sensitive and resistant cell lines were treated with dox (0.1, 0.25 uM) or vehicle control and apoptosis was determined by flow cytometry (Annexin-V) at 24 and 48 hrs. Cells were treated with dox or control for 24 hrs and western blotting was performed for mediators of apoptosis. Senescence was analyzed by ß-galactosidase staining following treatment with dox for 3-14 days. shRNA knockdown (KD) of p53 was performed in the CAL-51 (p53 WT cell line) and cells were subject to investigations above. Results: Doxorubicin treatment resulted in decreased cellular proliferation and increased apoptosis as assessed by Annexin-V expression in a subset of p53 WT and p53 Mut cell lines. Treatment with dox resulted in an increase in the pro-apoptotic proteins BID and p21, as well as a decrease in VAMP in sensitive cell lines. In a subset of cell lines resistant to dox treatment, we observed an increase in cells demonstrating phenotypic features of senescence and ß -galactosidase staining. We observed a decrease in p16 with dox treatment in the CAL-51 (p53 WT cell line) compared to an increase following treatment in p53 Mut cell lines. KD of p53 resulted in an increase in senescent cells following treatment with low dose dox. Conclusions: Treatment with doxorubicin resulted in different terminal cell phenotypes in TNBC cell lines with apoptosis observed in p53 WT and Mut cell lines. Senescence was observed in resistant cells and KD of p53 increased dox-induced senescence, confirming a role for p53 in mediating terminal cell fate. Efforts are ongoing to understand the role of mutant p53 in mediating terminal cell fate in response to dox and rational combinations to overcome dox-induced senescence may be clinically active in metastatic TNBC. Citation Format: Stephen G. Smoots, Anna R. Schreiber, Betelehem Yacob, Adrian Dominguez, Cecilia Levandowski, Todd M. Pitts, Jennifer R. Diamond. Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 136.
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Caccese, Mario, Tamara Ius, Matteo Simonelli, Matteo Fassan, Daniela Cesselli, Angelo Dipasquale, Francesco Cavallin, et al. "Mismatch-Repair Protein Expression in High-Grade Gliomas: A Large Retrospective Multicenter Study." International Journal of Molecular Sciences 21, no. 18 (September 14, 2020): 6716. http://dx.doi.org/10.3390/ijms21186716.
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Background: DNA mismatch repair (MMR) is a system for repairing errors in DNA replication. Cancer cells with MMR deficiency can have immunohistochemical loss of MMR protein expression leading to a hypermutable phenotype that may correlate with anti-PD1 efficacy. Scant data exist about immunohistochemical loss of MMR protein expression in high-grade gliomas (HGG). Materials and Methods: We performed a large multicenter retrospective study to investigate the frequency and the prognostic role of immunohistochemical loss of MMR protein expression in HGG patients; we nevertheless evaluated the association between this status and clinical or molecular characteristics. Immunohistochemical loss of MMR protein expression was recorded as partial or complete loss of at least 1 MMR protein. Results: We analyzed the expression of MMR proteins in tumor tissue of 355 consecutive patients. Partial and complete immunohistochemical loss of MMR proteins was found in 43/355 samples (12.1%) and among these, 15 cases (4.2%) showed a complete loss of at the least one MMR protein. Alteration of MSH2 expression was found in 55.8%, MSH6 in 46.5%, PMS2 in 34.9%, and MLH1 in 30.2%. Alteration of MMR protein expression was statistically more frequent in anaplastic gliomas, in recurrent disease, in patients treated with temozolomide, and in IDH-mut gliomas. Immunohistochemical loss of MMR proteins was not associated with survival, adjusting for clinically relevant confounders. Conclusions: MMR protein expression status did not affect survival in HGG patients. We identified clinical and molecular characteristics correlating with immunohistochemical loss of MMR proteins expression. A large study should be performed to analyze its predictive role of immune checkpoint inhibitor efficacy in these subgroups of patients.
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Ludwig, Nils, Aparna Rao, Saigopalakrishna Saileelaprasad Yerneni, Nduka Amankulor, and Theresa Whiteside. "Isolation and characterization of exosomes from IDH mutant gliomas." Journal of Clinical Oncology 37, no. 8_suppl (March 10, 2019): 152. http://dx.doi.org/10.1200/jco.2019.37.8_suppl.152.
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152 Background: Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are found in > 70% of intermediate grade gliomas. Gain-of-function mutations in IDH1 promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Tumor-derived exosomes (TEX) accumulate in the tumor microenvironment (TME) and in body fluids of patients. TEX serve as a communication system between tumor and normal cells. Circulating TEX carry a complex molecular and genetic cargo and interfere with functions of immune cells. This study characterizes TEX produced by IDH mutant glioma spheres and TEX from plasma of patients with IDH mutant gliomas. Methods: TEX produced by patient-derived IDH1(mut) or IDH1(wt) glioma spheres or TEX in patient’s plasma were isolated by mini size exclusion chromatography (mini-SEC). TEX morphology, size, numbers and molecular profile were characterized and the crossing of the blood-brain barrier (BBB) and biodistribution was investigated by in vivo imaging of TEX. Interactions of TEX with endothelial cells, astrocytes and immune cells were demonstrated by confocal microscopy. Results: TEX isolated from supernatants of IDH1(mut) or IDH1(wt) glioma spheres by mini-SEC carried TSG101 and showed the typical size and vesicular morphology of exosomes. BCA protein assays and qNano analysis revealed an elevated exosome production by IDH mutant cells ( p< 0.01). TEX carried immunosuppressive proteins (FasL, CTLA-4 and TRAIL) and IDH mutant exosomes carried higher levels of these proteins and of adenosine pathway components CD39 and CD73. Labeled TEX injected in the white matter of mice crossed the BBB and were detectable in distant organs 24h after injection. TEX were rapidly internalized by endothelial cells and astrocytes, but not by T cells, which interacted with TEX by surface signaling. These results were confirmed by investigating TEX isolated from glioma patient’s plasma. Conclusions: The data suggest that TEX play an important role in IDH mutant gliomas and drive tumor progression in part by inducing suppression of immune cells. Future efforts will focus on characterizing immunosuppressive effects of TEX derived from IDH mutant cells vs gliomas without IDH mutations.
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Volpe, Virginia Olivia, Nicole D. Vincelette, Onyee Chan, Chetasi Talati, Kendra Sweet, Andrew T. Kuykendall, Constantine Logothetis, et al. "High MYC Expression Predicts Poor Survival Outcomes in IDH1/2 Mutant AML Patients." Blood 138, Supplement 1 (November 5, 2021): 2377. http://dx.doi.org/10.1182/blood-2021-147030.
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Abstract Background In AML, IDH1/2 somatic mutations can be frequent in patients at about 20%. Physiologically, IDH1/2 catalyze isocitrate conversion to alpha-ketoglutarate (α-KG). α-KG is an activating co-factor for ten-eleven translocation 2 (TET2), an important epigenetic regulator which converts 5mC to 5hmC in the genomic DNA ultimately turning on the myeloid differentiation. In contrast, mutant IDH1/2 proteins promote α-KG conversion to 2-hydroxyglutarate (2-HG), an onco-metabolite. In pre-clinical studies, 2-HG inhibits TET2 activity by suppressing 5mC-to-5hmC conversion and thus allowing AML development and maintenance. Clinical trials of IDH1/2 inhibitors demonstrated promising outcomes in IDH1/2 mut AML patients. However, a substantial fraction of patients fail to respond to IDH1/2 inhibitors in AML. Our recent preclinical studies have shown MYC prevents the normal myeloid differentiation and cell death by suppressing IDH1/2-TET2 signaling. We evaluated if high MYC expression is associated with poor survival outcomes in IDH1/2 mut AML patients treated with Ivosidenib or Enasidenib Patients and Methods Patients diagnosed with AML at Moffitt Cancer Center between 2013-2020 were evaluated. IDH1/2 mutation status was assessed via next generation sequencing and those with documented IDH1/2 mut were included for the analysis. Clinical and demographic data were retrospectively extracted from the medical records. Pre-treatment MYC protein levels were assessed by immunohistochemistry staining using BM biopsy specimens. Statistical analyses were performed using GraphPad Prism (v.7.03) and SPSS (v.24.0). Baseline characteristics were compared by chi square (categorical variables) and t- test (continuous variables). Survival estimates were calculated using the Kaplan-Meier method from date of diagnosis and groups were compared using log-rank test. Multivariate survival analysis was performed by means of Cox proportional hazards regression Results A total of 28 (IDH1 mut, n=11; IDH2 mut, n=18; IDH1/2 mut, n=1) patients were included in the study. Median age at AML diagnosis was 66 years and 60% of patients were male. Twelve (42%) patients had secondary AML and 9 (32%), 11 (39%), and 6 (21%) patients had favorable, intermediate, and adverse risk, respectively, at AML diagnosis. A total of 17 (60.7%) and 6 (21.4%) patients received intensive chemotherapy and hypomethylating agents as their 1 st-line therapy. Among 28 patients, 22 (78.5%) and 6 (21.4%) patients had low and high MYC expression (≥5% by IHC staining), respectively, prior to IDH1/2 inhibitor treatment. Median number of treatments prior to IDH1/2 inhibitors was 3 (1-6) and the median duration of IDH1/2 inhibitor treatment was 3.2 (0.3-30) months (IDH1 mut, 3.3 [0.3-30] months; IDH2 mut, 2.5 [0.7-14.5] months). Treatment response was assessed in 27 patients and 11 (40.7%) had CR/CRi (high vs. low MYC, 20 vs. 45.5%, p=.6185). The median PFS was shorter in high MYC patients (1.6 vs. 4.4 months, HR=2.348, p=.0515). The median OS was also significantly shorter in high MYC patients (2.5 vs. 8.5 months, HR=5.971, p<.0001). Conclusion In our study, we demonstrated that high MYC expression was associated with significantly shorter PFS and OS in IDH1/2 mut AML patients. In consistent with our recent studies that showed MYC represses IDH1/2 and TET2 expression in AML, this study suggests that MYC may play an important role in the resistance mechanism of IDH1/2 inhibitors. Additional studies with larger cohorts are warranted to further confirm and validate these findings. Disclosures Talati: Jazz: Speakers Bureau; Astellas: Speakers Bureau; BMS: Honoraria; AbbVie: Honoraria; Pfizer: Honoraria. Sweet: Bristol Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AROG: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kuykendall: Pharmaessentia: Honoraria; Celgene/BMS: Honoraria; Incyte: Consultancy; Abbvie: Honoraria; Blueprint: Honoraria; Novartis: Honoraria, Speakers Bureau; Protagonist: Consultancy, Research Funding. Komrokji: Geron: Honoraria; Acceleron: Honoraria; Novartis: Honoraria; Agios: Honoraria, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; JAZZ: Honoraria, Speakers Bureau. Sallman: Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Intellia: Membership on an entity's Board of Directors or advisory committees; Magenta: Consultancy; Syndax: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Takeda: Consultancy; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding. Padron: Kura: Research Funding; Incyte: Research Funding; BMS: Research Funding; Stemline: Honoraria; Blueprint: Honoraria; Taiho: Honoraria.
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McCubrey, James A., Li V. Yang, Stephen L. Abrams, Linda S. Steelman, Matilde Y. Follo, Lucio Cocco, Stefano Ratti, Alberto M. Martelli, Giuseppa Augello, and Melchiorre Cervello. "Effects of TP53 Mutations and miRs on Immune Responses in the Tumor Microenvironment Important in Pancreatic Cancer Progression." Cells 11, no. 14 (July 9, 2022): 2155. http://dx.doi.org/10.3390/cells11142155.
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Approximately 90% of pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). PDAC is the fourth leading cause of cancer death world-wide. Therapies for PDAC are largely ineffective due to the dense desmoplastic tumor microenvironment which prevents chemotherapeutic drugs and small molecule inhibitors from exerting effective anti-cancer effects. In this review, we will discuss the roles of TP53 and miRs on the PDAC tumor microenvironment and how loss of the normal functions of TP53 promote tumor progression. The TP53 gene is mutated in approximately 50% of pancreatic cancers. Often, these TP53 mutations are point mutations which confer additional functions for the TP53 proteins. These are called gain of function (GOF) mutations (mut). Another class of TP53 mutations are deletions which result in loss of the TP53 protein; these are referred to TP53-null mutations. We have organized this review into various components/properties of the PDAC microenvironment and how they may be altered in the presence of mutant TP53 and loss of certain miR expression.
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Key, Jana, Sylvia Torres-Odio, Nina C. Bach, Suzana Gispert, Gabriele Koepf, Marina Reichlmeir, A. Phillip West, et al. "Inactivity of Peptidase ClpP Causes Primary Accumulation of Mitochondrial Disaggregase ClpX with Its Interacting Nucleoid Proteins, and of mtDNA." Cells 10, no. 12 (November 29, 2021): 3354. http://dx.doi.org/10.3390/cells10123354.
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Biallelic pathogenic variants in CLPP, encoding mitochondrial matrix peptidase ClpP, cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are the main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brains. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with the nucleoid component POLDIP2, the mitochondrial poly(A) mRNA granule element LRPPRC, and tRNA processing factor GFM1 (in mouse, also GRSF1). Only in mouse did accumulated ClpX, GFM1, and GRSF1 appear in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely, OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2, and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids and show nucleoid enlargement in human as a key consequence.
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Gómez, Carmen Elena, Beatriz Perdiguero, Michela Falqui, María Q. Marín, Martina Bécares, Carlos Óscar S. Sorzano, Juan García-Arriaza, Mariano Esteban, and Susana Guerra. "Enhancement of HIV-1 Env-Specific CD8 T Cell Responses Using Interferon-Stimulated Gene 15 as an Immune Adjuvant." Journal of Virology 95, no. 2 (October 28, 2020): e01155-20. http://dx.doi.org/10.1128/jvi.01155-20.
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ABSTRACTInduction of the endogenous innate immune system by interferon (IFN) triggers the expression of many proteins that serve like alarm bells in the body, activating an immune response. After a viral infection, one of the genes activated by IFN induction is the IFN-stimulated gene 15 (ISG15), which encodes a ubiquitin-like protein that undergoes a reversible posttranslational modification (ISGylation). ISG15 protein can also act unconjugated, intracellularly and secreted, acting as a cytokine. Although ISG15 has an essential role in host defense responses to microbial infection, its role as an immunomodulator in the vaccine field remains to be defined. In this investigation, we showed that ISG15 exerts an immunomodulatory role in human immunodeficiency virus (HIV) vaccines. In mice, after priming with a DNA-ISG15 vector mixed with a DNA expressing HIV-1 gp120 (DNA-gp120), followed by a booster with a modified vaccinia virus Ankara (MVA) vector expressing HIV-1 antigens, both wild-type ISG15-conjugated (ISG15-wt) and mutant unconjugated (ISG15-mut) proteins act as immune adjuvants by increasing the magnitude and quality of HIV-1-specific CD8 T cells, with ISG15-wt providing better immunostimulatory activity than ISG15-mut. The HIV-1 Env-specific CD8 T cell responses showed a predominant T effector memory (TEM) phenotype in all groups. Moreover, the amount of DNA-gp120 used to immunize mice could be reduced 5-fold after mixing with DNA-ISG15 without affecting the potency and the quality of the HIV-1 Env-specific immune responses. Our study clearly highlights the potential use of the IFN-induced ISG15 protein as immune adjuvant to enhance immune responses to HIV antigens, suggesting that this molecule might be exploitable for prophylactic and therapeutic vaccine approaches against pathogens.IMPORTANCE Our study described the potential role of ISG15 as an immunomodulatory molecule in the optimization of HIV/AIDS vaccine candidates. Using a DNA prime–MVA boost immunization protocol, our results indicated an increase in the potency and the quality of the HIV-1 Env-specific CD8 T cell response. These results highlight the adjuvant potency of ISG15 to elicit improved viral antigen presentation to the immune system, resulting in an enhanced HIV-1 vaccine immune response. The DNA-ISG15 vector could find applicability in the vaccine field in combination with other nucleic acid-based vector vaccines.
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Kang, Nan, Zhiqi Wang, Fei Gai, Wenqing Su, Danhua Shen, and Jianliu Wang. "Molecular classification of endometrial cancer of Chinese population." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): e17623-e17623. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e17623.
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e17623 Background: Endometrial cancer (EC) is one of the most prevalent gynecologic tumors. Current diagnosis and treatment of EC no longer rely solely on traditional histopathological classification. Nevertheless, molecular classification of EC demonstrated clear prognostic value and may guide clinical decision. Methods: In this study, archived tissue specimens from 240 EC patients from Department of Pathology, Peking University People’s Hospital. Four subtypes [POLE ultramutated (POLE mut), microsatellite instability high (MSI-H), copy number low (CNL), and copy number high (CNH)] were stratified by next-generation sequencing (NGS) panel (Amoy Diagnostics, Xiamen, China) targeting POLE, TP53, BRCA1, and BRCA2 genes and microsatellite instability (MSI) status. Immunohistochemistry (IHC) was applied to detect the expression of P53, MMR and other related proteins. Results: Distribution of the EC subtypes in 240 patients was 13 (5.42%) of POLE mut, 36 (15.00%) of MSI-H, 41 (17.08%) of CNH, and 150 (62.50%) of CNL. Compared to published results of EC subtypes in Caucasian including TCGA, ProMisE as well as TransPORTEC, real-world data on Chinese ECs displayed a significantly larger proportion of CNL. In addition, novel biomarkers such as DUSP1, MCF7 and BUB1, which were independent prognostic marker from our previous research were validated. Also, it was found that BRCA2 appeared to be more prevalent in EC than BRCA1. Further analysis revealed that the overall consistency for NGS-based and IHC-based TP53 abnormalities detection and MSI/MMR status assessment were as high as 87.5% and 100%, respectively. Conclusions: Chinese ECs have unique molecular characteristics. In order to perform accurate molecular typing of Chinese ECs, more molecular indicators that match the characteristics of the Chinese population need to be added to the existing classifiers. NGS-based panel is easy to operate and replicate with high accuracy. Thus, it is a viable alternative to IHC in EC molecular classification.
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Farkas, Tamas, Joerg Meerpohl, Shinsuke Hirabayashi, Alyson MacInnes, Gunda Ruzaike, Paul Essers, Sandra Urbaniak, et al. "Characteristics of Diamond Blackfan Anemia Patients with Unknown Genetic Defect." Blood 120, no. 21 (November 16, 2012): 1267. http://dx.doi.org/10.1182/blood.v120.21.1267.1267.
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Abstract Abstract 1267 Diamond Blackfan anemia (DBA) has been established as a ribosomopathy and results in the majority of cases from the haploinsufficiency of genes coding for ribosomal proteins (RP). So far, 12 cytoplasmic RP genes (S7, S10, S17, S19, S24, S26, L5, L9, L11, L19, L26, and L35a) and the transcription factor GATA1 were identified to be affected in approximately half of all patients. Additionally, RP-gene deletions might constitute the causative mechanism. From the patients currently enrolled in the DBA registry in Germany, material for extensive genetic studies was available in 186 cases. We identified mutations in 52% (n=96) of patients in 9 of the before-mentioned 12 RP-genes. In addition, we investigated GATA1 and KLF1, a key erythroid transcription factor associated with the persistence of high levels of fetal hemoglobin (HbF), but no mutations in DBA patients were found. We next focused on the identification of RP-gene deletions within the mutation-negative cohort. Using Affymetrix 6.0 SNP-arrays and a qPCR-based copy number assay, we identified RP-gene deletions in 10% (n=19) patients, with S17 being the most frequently deleted gene in 6 patients, followed by L5, L11, L35a, and S19. To assess whether the not yet identified genetic defects in the remaining ∼1/3 negative patients might have a different impact on the physical development and the clinical course than the RP-genes already linked to DBA, we compared both mutation/deletion positive (mut/del+) and negative cohorts. The age at diagnosis of mut/del+ patients versus negative patient group was 0.3 years as compared to 0.7 years, and there was equal sex ratio in both groups. HbF and eADA, used as lab surrogate markers, were increased in 73% mut/del+ as compared to 75% negative cases. The prevalence of physical abnormalities between both groups also seems to be similar. Specifically, no difference was seen between mut/del+ and negative patients for heart defects (37.5% and 26.5%), urogenital malformations (6.7% and 8.8%), thumb malformations (11.5% and 5.9%), small for gestational age (17% and 10.5%) and craniofacial malformations (39.4% and 35.3%). However cleft palate was exclusive to only one of 68 evaluable negative patients as compared to 6 of 104 evaluable mut/del+ positive patients. Interestingly, nearly all patients (94%) with gene deletions presented with physical anomalies, which might result from haploinsufficiency of other genes affected in the context of a continuous gene syndrome. We next focused our analysis on treatment modalities. Comparing the rate of spontaneous remission (SR), there were significantly more patients with SR within negative patient group as compared to mut/del+ cohort: 31.6% (18 of 57 evaluable) vs. 15.2% (15 of 99 evaluable), p < 0.04. Contrasting this, there was no difference for other treatment modalities including steroid/transfusion dependency. In an attempt to identify whether the patients with an unknown genetic defect have impaired ribosome biogenesis, we performed polysomal profiling of 3 subgroups of DBA patients, those with point mutations in known RP genes, those with large deletions spanning known RP genes, and those that were deletion and mutation negative. Our preliminary results obtained from a cohort of 30 patients cell lines revealed not only the expected imbalance in 40S to 60S peak size depending on which RP gene was mutated, but also the presence of ribosomal intermediate structures in negative patients which were previously not reported in context of DBA. Some of these profiles with intermediates are linked to RP genes that have known links to rRNA processing. Interestingly, some of the profiles of the known RP genes were recapitulated in the profiles from patients with unknown genetic defect, suggesting that ribosomal profiling may be a valuable method of prediction for future DBA genotyping efforts. In summary, DBA patients with unkown genetic defect show similar clinical presentation as patients with known RP-gene mutations or deletions. The observation of different treatment outcomes requires further investigation. Polysome profilling provides initial suggestion about the type of RP-defect and can help in better characterizing the patients prior to prospective next generation sequencing - based gene discovery. Disclosures: No relevant conflicts of interest to declare.
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Li, Ling, Zijun Yidan Xu-Monette, Chi Young Ok, Alexander Tzanko, Carlo Visco, Nora Gisin, Santiago Montes-Moreno, et al. "NF-κB Subunit c-Rel Cooperates with Myc and Mutated p53 to Confer Significantly Worse Survival in Patients with Diffuse Large B-Cell Lymphoma: A Report from the International DLBCL Rituximab-CHOP Consortium Program." Blood 124, no. 21 (December 6, 2014): 1620. http://dx.doi.org/10.1182/blood.v124.21.1620.1620.
Full textAbstract:
Abstract Objective :To evaluate the prognostic significance of RELamplification, expression and c-Rel activation in DLBCL patients and to identify potential mechanisms for impact of c-Rel activation on patient survival. Patients and Design : The study cohort consists 460 de novo DLBCL patients (median follow-up, 46.8 moths) treated with R-CHOP. We assessed the nuclear expression/activation of c-Rel and other NF-κB subunits by immunohistochemistry, REL gene amplification by fluorescence in situ hybridization, and gene expression profiling using Affymetrix GeneChips array. Correlations between expression of nuclear c-Rel, REL mRNA, and expression of TP53, MDM2, MDM4, MYC, BCL2, AKT1, NFKB1, RELA, NFKB2, and RELB, both at the mRNA and protein levels were analyzed using t-tests. The prognostic significance of c-Rel activation, REL mRNA expression, and REL amplification was evaluated in the overall cohort, and different subgroups stratified by COO, status of TP53 mutation (wide type/WT, or mutated/MUT) and expression, Myc, Bcl-2 overexpression, and nuclear expression of other NF-κB subunits. Results :Nuclear c-Rel expression was observed in 29.6% of DLBCL patients and did not correlate with REL mRNA levels (P=0.95) and COO (P=0.77). In contrast, REL mRNA was significantly higher in germinal center B-like (GCB) subtype (P<0.0001). In GCB-DLBCL, nuclear c-Rel expression was associated with significantly lower Myc, p53, MDM4, and pAKT protein levels but not at the transcriptional level. In contrast, in ABC-DLBCL, c-Rel activation was associated with significantly higher MUT-TP53mRNA level and reduced pAKT expression. Correspondingly to the lack of associations with reduced Myc, pAKT, and p53 in ABC-DLBCL, nuclear c-Rel expression showed prognostic impact only in ABC- but not in GCB- DLBCL, especially when it was concurrent with Myc overexpression (P<0.0001 for OS and P=0.0012 for PFS). Importantly, patients with c-Rel activation and p53 mutations had significantly worse survival (for OS, hazard ratio, 3.56; P=0.0011; median survival, 16.2 vs 87.3 months. For PFS, hazard ratio, 3.976; P=0.0004; median survival, 10.4 vs 55.5 months) compared with other MUT-p53 DLBCL patients. The additive impact of c-Rel activation to TP53 mutations was more apparent in the ABC-DLBCL subtype, in which c-Rel activation was associated with significantly upregulated MUT-TP53 transcription (P=0.0087). Conversely, MUT-p53 expression was associated significantly with upregulated REL mRNA expression (P=0.0021), predominantly in the ABC-DLBCL subtype (P=0.0047). Only in ABC-DLBCL patients, TP53 mutations were associated with elevated nuclear c-Rel levels with a borderline P value (P=0.05). In addition to the association of nuclear p65 in GCB-DLBCL (P=0.003), and p50, NFKB1 and RELA mRNA in ABC-DLBCL (P=0.0023), the prognostic significance of c-Rel appears to depend on p50 (P=0.08) and p65 expression (P=0.12). Also, supporting that c-Rel transactivates anti-apoptotic BCL2L1/BCLXL by previous studies, there was no significant difference in survival of ABC-DLBCL patients with isolated BCL2 overexpression and with nuclear c-Rel expression. Comprehensive gene-expression profiling analysis and cell line study are undergoing in order to identify pathways to activate c-Rel and oncogenic mechanisms for c-Rel–mediated chemoresistance. REL amplification was predominantly observed in GCB-DLBCL (frequency, 7%) and correlated with significantly higher mRNA level (P<0.0001). However, REL amplification did not correlation with nuclear c-Rel expression or patient survival, illustrating the importance of posttranslational regulations in c-Rel activation and function. Consistent with the adverse impact of c-Rel activation at the protein level, a strong trend toward poor survival was observed for elevated RELmRNA in ABC- but not in GCB-DLBCL. Conclusions : Nuclear c-Rel activation was associated with reduced level of proteins whose degradation involves with ubiquitin-proteasome in GCB-DLBCL, and upregulated TP53 transcription in ABC-DLBCL. Reciprocal regulation of c-Rel and MUT-p53 at the transcriptional level may underlie the synergetic adverse effect of c-Rel activation and TP53 mutations. c-Rel cooperated with Myc and conferred significantly worse survival in ABC-DLBCL patients. These subsets of c-Rel+ DLBCL patients likely will benefit from c-Rel targeted therapies. Disclosures No relevant conflicts of interest to declare.